Glucose Determination in Beverages Using Carbon Nanotube

Laboratory Experiment pubs.acs.org/jchemeduc

Glucose Determination in Beverages Using Carbon Nanotube Modified Biosensor: An Experiment for the Undergraduate Laboratory J. Mark Hobbs,† Niral N. Patel,†,§ Daniel W. Kim,†,# Joseph K. Rugutt,‡ and Adam K. Wanekaya*,† †

Department of Chemistry, Missouri State University, Springfield, Missouri 65897, United States Department of Chemistry, Missouri State University−West Plains, Garfield, Missouri 65775, United States

‡

S Supporting Information *

ABSTRACT: We describe a versatile method for students to fabricate carbon nanotube-based enzyme-modified biosensor electrodes using a layerby-layer electrostatic self-assembly procedure. Amperometric experiments employing a simple three-electrode cell enabled sensitive and selective determination of glucose in various commercially produced beverages familiar to students. The method was optimized with respect to various parameters, and the results compared very well with standard methods used for glucose determination. The procedure is versatile, robust, and relatively inexpensive. It can be performed by undergraduate students as was demonstrated by the good results obtained by the upper-level instrumental analysis class. The experiment can easily be integrated into laboratory classes for analytical chemistry, biotechnology, or biochemistry students to demonstrate important principles and techniques of nanoscale science, materials science, biochemistry, electrochemistry, and sensor technology. KEYWORDS: Second-Year Undergraduate, Upper-Division Undergraduate, Analytical Chemistry, Laboratory Instruction, Hands-On Learning/Manipulatives, Bioanalytical Chemistry, Biotechnology, Electrochemistry, Food Science Nanotechnology

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determination of glucose in various beverages. Conventional electrodes are modified with carbon nanotubes and glucose oxidase enzyme via a layer-by-layer self-assembly procedure involving the electrostatic interactions between the negatively charged enzyme layer and a positively charged polymer layer.1j,4 The procedure is simple, versatile, relatively inexpensive, and easily integrated into laboratory classes to demonstrate important principles and techniques of nanoscale science, materials science, biochemistry, electrochemistry, and sensor technology. The experiment therefore offers training in areas that are sometimes underemphasized, in addition to building instrumental analysis skills. The suitability of carbon nanotubes for sensing applications is due to their electrocatalytic and electrochemical sensitivity, unique properties such as increased electrode surface area,5 fast electron transfer rate,6 significant mechanical strength, and good chemical stability.7 Past studies have demonstrated that carbon nanotubes (CNTs) impart strong electrocatalytic activity and minimize electrode fouling.8 The electrocatalytic effect of CNTs has been attributed to the activity of the edgeplane-like graphite sites at the CNTs ends.9 Thus, the use of electron mediators is not required. A well-known glucose assay is adopted that uses glucose oxidase, GOx, to catalyze the

anotechnology has generated a lot of interest because nanoscale materials (having dimensions of roughly 1− 100 nm) exhibit unique properties that enable their use in novel applications. The incorporation of nanotechnology and nanotechnology-related classes and laboratories in the undergraduate college curriculum should expose students to exciting, cutting-edge research and prepare them for future careers in nanotechnology-related and other fields. Glucose determination has important applications in clinical chemistry and food science. Although numerous publications involving glucose determination can be found in the literature,1 very few2 have been adapted for laboratory teaching to demonstrate their analytical utility. In fact, among the previous laboratories and experiments published in this Journal2,3 about glucose detection, only one2 used nanoscale materials. Here, we describe a simple method for students to fabricate carbon nanotube−enzyme-modified amperometric biosensor electrodes for determination of glucose in beverages. Students thus have the opportunity to experience the power of nanotechnology and electrochemistry and explore concepts not commonly addressed in undergraduate chemistry laboratories. Previous laboratories about glucose detection have mainly been performed without the use of nanoscale materials.3 As a result, most of them use mediators to facilitate efficient electron transfer. In this approach, amperometric experiments using a common three-electrode cell enable sensitive and selective © XXXX American Chemical Society and Division of Chemical Education, Inc.

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dx.doi.org/10.1021/ed300429a | J. Chem. Educ. XXXX, XXX, XXX−XXX

Journal of Chemical Education

Laboratory Experiment

Lafayette, IN). A steel sewing needle or wire can be used in instead of the platinum wire in this experiment. All potentials were referenced to Ag/AgCl. A conventional ultrasonicator was used to sonicate the nanotubes and a magnetic stirrer and stir bar were used to agitate the sample during electrochemical measurements. Details of reagent preparation are described in the Supporting Information section.

oxidation of glucose in presence of dissolved oxygen, producing β-gluconic acid and hydrogen peroxide (eq 1). GOX

β ‐glucose + O2 + H 2O ⎯⎯⎯⎯→ β ‐gluconic acid + H 2O2 (1)

The quantity of glucose present in solution is directly proportional to the quantity of H2O2 produced. Using a simple potentiostat, the H2O2 is electrochemically oxidized at +0.8 V (vs Ag/AgCl, eq 2), H 2O2 → O2 + 2H+ + 2e−

Electrode Preparation and Fabrication

The students can either work individually or in pairs. The students fabricated the electrode as follows. In a typical experiment, glassy carbon electrodes (GCEs) were polished using 1.0 μm alumina followed by 0.05 μm alumina, and rinsed with deionized water. A small volume of the MWNT−COOH suspension, 10 μL, was pipetted onto the surface of each GCE. The GCEs were allowed to dry at 100 °C for 10 min and were then cooled. A small volume of the PDDA solution, 10 μL, was then pipetted on the tip of each GCE and dried at 100 °C for 10 min. After rinsing by suspension in deionized water, a layer of PDDA remained electrostatically attached to the negatively charged MWNT−COOH surface of the GCEs because the PDDA solution carries a net positive charge. The buffered GOx solution, 10 μL, was then pipetted onto the tip of each MWNT−COOH−PDDA electrode and dried at 100 °C for 10 min. GOx has an isoelectric point of approximately 4.5, so it carries a net negative charge in this solution. Therefore, after being rinsed as above, a layer of GOx remained electrostatically attached to the positively charged PDDA. Ideally, the drying of the electrode after casting with the enzyme should be done at ambient conditions due to the potential of enzyme denaturation. However, this would require too much time considering the time allowed for most undergraduate labs. We found that drying the electrode at 100 °C for 10 min did not result in any noticeable change in the activity of the enzyme. Finally, 10 μL of the PDDA solution was pipetted again onto the tip of each MWNT−COOH−PDDA−GOx electrode, dried at 100 °C for 10 min, and rinsed by suspension in deionized water as above. A layer of positively charged PDDA remained electrostatically attached to the negatively charged GOx layer beneath. The GOx was thus immobilized between two layers of PDDA (Figure 1).

(2)

resulting in a current, i, i=

nFADC δ

(3)

whose magnitude directly proportional to n, the number of electrons involved in the oxidation process; F, Faraday’s constant; A, the active surface area of the electrode; D, the diffusion coefficient; C, the bulk concentration; and inversely proportional to δ, the thickness of the diffusion layer. Before the experiments, students are made aware of the advantages of biosensors such as fast response, simple design, low cost, little or no power requirement, and ease of use and interpretation. Students should also be made aware of their potential limitations such as long-term stability issues and variability in signal intensity. Nevertheless, their successful use in point-of-care and point-of-use scenarios should also be highlighted. For example, home-based glucose test devices are just one example of successful electrochemical devices. The market of these devices is in excess of $6 billion10 and growing due to aging population, unhealthy lifestyle, and rise in obesity related issues.

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EXPERIMENTAL DETAILS

Equipment, Chemicals, and Reagents

Multiwalled carbon nanotubes, 30 nm, (NanoLab Inc., Waltham, MA) in 3:1 sulfuric−nitric acid were sonicated for 4 h, vacuum filtered, washed with deionized water until acid free, and dried at 100 °C for 12 h. The oxidized multiwalled nanotubes (MWNT−COOH) were then suspended in dimethyl formamide at a concentration of 5 mg/mL. This nanotube suspension can be used for years. The suspension required sonication for 1 to 2 min prior to use. Each student group requires 20 μL of this suspension. A solution of poly(diallyldimethylammonium chloride) (PDDA, Aldrich, St. Louis, MO) was prepared in 0.5 M NaCl at a concentration of 1 mg/mL. Phosphate buffer was prepared at a concentration of 0.1 M and pH of 8.0. Type VII glucose oxidase (GOx, Aldrich, St. Louis, MO) solution was prepared by adding glucose oxidase to pH 8.0 phosphate buffer prepared above. Final concentration of the solution was 20 mg of the enzyme/mL of buffer. This solution was refrigerated to preserve enzymatic activity. The instructor ensured that the above reagents and chemicals were available to the students prior to start of lab session. Equipment used in developing this experiment included a Princeton Applied Research VMP3 potentiostat controlled by EC-Labs software (Biologic, Knoxville, MA). Other types of potentiostats may be used. Glassy carbon working electrodes, 3 mm diameter, Ag/AgCl reference electrode, alumina polish, polishing pads, platinum wire counter electrode, and electrochemical cell vial were obtained from BASi Corp (West

Electrochemical Experiments

Once the GCEs are prepared, amperometric experiments were performed at a constant potential of +0.8 V using a simple

Figure 1. Layer-by-layer electrostatic assembly. A layer of glucose oxidase immobilized between layers of poly(diallyldimethylammonium chloride) (GCE = glassy carbon electrode; MWNT−COO− = carboxylated multiwalled carbon nanotubes; PDDA = poly(diallyldimethylammonium chloride; GOx = glucose oxidase). B




three-electrode arrangement suspended in buffer solution (Figure 2). The modified GCE was used as the working

Figure 3. Typical amperogram generated by students showing increase of current vs time upon addition of glucose. Arrows correspond to points where the concentration of glucose in the working solution was increased by 0.5 mM. The amperogram here shows data acquisition after an initial sharp increase in current followed by an exponential decay to a stable current (at about 600 s). Subsequent aliquots of glucose solution are the added at about 60 s intervals. Each run takes less than 15 min.

Figure 2. Three-electrode electrochemical cell setup with buffer solution.

electrode with a platinum wire as the counter electrode and Ag/ AgCl as the reference electrode. Amperometric experiments were performed by applying a potential of 0.8 V vs Ag/AgCl and monitoring the current as aliquots of standard solution were added sequentially. The solution was magnetically stirred to increase mass transport of the analyte to the electrode surface.

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HAZARDS Dimethyl formamide is a high boiling point flammable liquid. It is harmful if absorbed through skin. PDDA and carbon nanotubes can be irritating. This experiment contains no other significant hazards. Safety goggles are required and lab coats are recommended.

Figure 4. Typical calibration curve for total current vs glucose standard concentration generated by students.

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RESULTS AND DISCUSSION A typical amperogram resulting from the addition of glucose to the above electrochemical cell is shown in Figure 3. Initially, the current is allowed stabilize on application of +0.8 V. This normally takes a couple of minutes. On addition of the first aliquot of glucose (point a), there is an instantaneous increase in the current resulting from the oxidation of H2O2 produced from the catalysis of glucose oxidation by GOx immobilized on the electrode surface (eq 1). The increase in current should only be observed when glucose is added to the electrochemical cell as it is the only substance whose oxidation will be catalyzed by glucose oxidase. This enables the biosensor to exhibit excellent selectivity. On the other hand, the magnitude of the current is proportional to the concentration of glucose in the electrochemical cell. The biosensor attains a steady-state current within 60 s after the initial addition of glucose. More aliquots of glucose solution were added at points b, c, and d with more or less the same result. A typical calibration plot of total current versus the increase in glucose concentration is shown in Figure 4. The plot is linear with a squared correlation coefficient of 0.999. After calibration, the determination of glucose in various beverages was attempted.

Aliquots of beverages were introduced into the electrochemical cell without any prior sample treatment. The magnitude of the resulting current was noted and glucose concentration was interpolated from the calibration plot. Students in the lab performed the determination of glucose in Sprite and found it to be 0.253 ± 0.053 M. Because “sugars” rather than glucose is listed in the nutritional information of most beverages, glucose determination was performed using an officially accepted standard optical method. Using this method the glucose content was found to be 0.264 ± 0.036 M.11 This demonstrates the versatility and accuracy of the glucose biosensor. In general, the variability between student groups was less than 14% which is not unusual for enzyme electrodebased sensors.12 This indicates the potential use of this experiment for teaching purposes. A variant of this soft drink, sweetened with aspartame and acesulfame potassium rather than high fructose corn syrup, is marketed under the brand Sprite Zero. The glucose biosensor was used to determine the glucose in this beverage. From an aliquot of this product, the glucose concentration was undetectable. C



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Colorimetric detection of glucose in rat brain using gold nanoparticles. Angew. Chem., Int. Ed. 2010, 49 (28), 4800−4804. (d) Jin, L.; Shang, L.; Guo, S.; Fang, Y.; Wen, D.; Wang, L.; Yin, J.; Dong, S. Biomolecule-stabilized Au nanoclusters as a fluorescence probe for sensitive detection of glucose. Biosens. Bioelectron. 2011, 26 (5), 1965− 1969. (e) Radhakumary, C.; Sreenivasan, K. Naked eye detection of glucose in urine using glucose oxidase immobilized gold nanoparticles. Anal. Chem. 2011, 83 (7), 2829−2833. (f) Rassaei, L.; Marken, F. Pulse-voltammetric glucose detection at gold junction electrodes. Anal. Chem. 2010, 82 (17), 7063−7067. (g) Song, Y.; Qu, K.; Zhao, C.; Ren, J.; Qu, X. Graphene oxide: Intrinsic peroxidase catalytic activity and its application to glucose detection. Adv. Mater. 2010, 22 (19), 2206− 2210. (h) Wu, P.; Shao, Q.; Hu, Y.; Jin, J.; Yin, Y.; Zhang, H.; Cai, C. Direct electrochemistry of glucose oxidase assembled on graphene and application to glucose detection. Electrochim. Acta 2010, 55 (28), 8606−8614. (i) Xie, J.; Huang, Y. Co3O4 nanoparticles-enhanced luminol chemiluminescence and its application in H2O2 and glucose detection. Anal. Methods 2011, 3 (5), 1149−1155. (j) Liu, G.; Lin, Y. Amperometric glucose biosensor based on self-assembling glucose oxidase on carbon nanotubes. Electrochem. Commun. 2006, 8 (2), 251−256. (2) Bai, J.; Flowers, K.; Benegal, S.; Calizo, M.; Patel, V.; Bishnoi, S. W. Using the Enzymatic Growth of Nanoparticles To Create a Biosensor. An Undergraduate Quantitative Analysis Experiment. J. Chem. Educ. 2009, 86 (6), 712. (3) (a) Blanco-López, M. C.; Lobo-Castañoń , M. J.; MirandaOrdieres, A. J. Homemade Bienzymatic-Amperometric Biosensor for Beverages Analysis. J. Chem. Educ. 2007, 84 (4), 677. (b) Gooding, J. J.; Yang, W.; Situmorang, M. Bioanalytical Experiments for the Undergraduate Laboratory: Monitoring Glucose in Sports Drinks. J. Chem. Educ. 2001, 78 (6), 788. (c) Sadik, O. A.; Brenda, S.; Joasil, P.; Lord, J. Electropolymerized Conducting Polymers as Glucose Sensors. J. Chem. Educ. 1999, 76 (7), 967. (d) Wang, J.; Macca, C. Use of Blood-Glucose Test Strips for Introducing Enzyme Electrodes and Modern Biosensors. J. Chem. Educ. 1996, 73 (8), 797. (4) (a) Decher, G. Fuzzy Nanoassemblies: Toward Layered Polymeric Multicomposites. Science 1997, 277 (5330), 1232−1237. (b) Wang, J.; Liu, G.; Lin, Y. Layer-by-Layer Assembly of Enzymes on Carbon Nanotubes. In Biomolecular Catalysis; American Chemical Society: Washington, DC, 2008; Vol. 986, pp 117−128. (5) Peigney, A.; Laurent, C.; Flahaut, E.; Bacsa, R. R.; Rousset, A. Specific surface area of carbon nanotubes and bundles of carbon nanotubes. Carbon 2001, 39 (4), 507−514. (6) Gooding, J. J.; Wibowo, R.; Liu, J. Q.; Yang, W. R.; Losic, D.; Orbons, S.; Mearns, F. J.; Shapter, J. G.; Hibbert, D. B. Protein electrochemistry using aligned carbon nanotube arrays. J. Am. Chem. Soc. 2003, 125 (30), 9006−9007. (7) (a) Treacy, M. M. J.; Ebbesen, T. W.; Gibson, J. M. Exceptionally high Young’s modulus observed for individual carbon nanotubes. Nature 1996, 381 (6584), 678−680. (b) Ajayan, P. M. Nanotubes from carbon. Chem. Rev. 1999, 99 (7), 1787−1799. (8) (a) Wang, J.; Musameh, M.; Lin, Y. H. Solubilization of carbon nanotubes by Nafion toward the preparation of amperometric biosensors. J. Am. Chem. Soc. 2003, 125 (9), 2408−2409. (b) Musameh, M.; Wang, J.; Merkoci, A.; Lin, Y. H. Low-potential stable NADH detection at carbon-nanotube-modified glassy carbon electrodes. Electrochem. Commun. 2002, 4 (10), 743−746. (c) Wang, J. X.; Li, M. X.; Shi, Z. J.; Li, N. Q.; Gu, Z. N. Direct electrochemistry of cytochrome c at a glassy carbon electrode modified with single-wall carbon nanotubes. Anal. Chem. 2002, 74 (9), 1993−1997. (9) Liu, G. D.; Riechers, S. L.; Mellen, M. C.; Lin, Y. H. Sensitive electrochemical detection of enzymatically generated thiocholine at carbon nanotube modified glassy carbon electrode. Electrochem. Commun. 2005, 7 (11), 1163−1169. (10) Global Industry Analysts, I. Biosensors in Medical Diagnostics A Global Strategic Bussiness Report; February 2012. (11) The authors carried out the glucose determination using an officially accepted standard optical method to validate the technique. It is not necessary to do this to run the experiment.

CONCLUSIONS Glucose determination was carried out using conventional glassy carbon electrodes modified with carbon nanotubes and glucose oxidase using a layer-by-layer procedure. Amperometric experiments employing a simple three-electrode cell enabled sensitive and selective determination of glucose in commercially produced sugary beverages familiar to students. The procedure is robust, simple, versatile, and relatively inexpensive, and it can be performed by undergraduate students using a simple potentiostat. Most of the reagents and equipment are found in a typical chemistry or biochemistry lab. Pristine high quality unfunctionalized carbon nanotubes are available commercially at an average cost of $100/g. A 5 mg/mL suspension of the oxidized nanotubes in dimethyl formamide can fabricate more than 50 electrodes. The experiment has been tried, tested, and found suitable for testing glucose content in soft drink beverages. Depending on the quality of electrode fabrication, the linear range may go up to 15 mM. The electrode response did not significantly change three months after fabrication when stored at 4 °C. Instructors are advised to test the applicability of this technique to other types of beverages. The experiment is a good example of an intersection of nanotechnology, electrochemistry, analytical chemistry, and biochemistry. It can easily be integrated into laboratory classes for chemistry, biotechnology, or biochemistry students to demonstrate important principles and techniques of nanoscale science, materials science, biochemistry, electrochemistry, and sensor technology.

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ASSOCIATED CONTENT

S Supporting Information *

Notes for instructors and instructions for students. This material is available via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Present Addresses §

Niral N. Patel: Division of Pediatric Endocrinology and Diabetes, Vanderbilt University, Nashville, Tennessee 37232, United States. # Daniel W. Kim: Harvard University Medical School, Boston, Massachusetts 02115, United States. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors wish to acknowledge Summer Undergraduate Research Fellowship funding from the Department of Chemistry at Missouri State University to support Daniel Kim. The following undergraduate students are also acknowledged for participating in actual lab experiments: Ryan Aubuchon, Michelle Butts, Joshua Holland, Stephanie Klamm, Sarah Robinson, and Aaron Simpson.

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REFERENCES

(1) (a) Ding, Y.; Wang, Y.; Su, L.; Bellagamba, M.; Zhang, H.; Lei, Y. Electrospun Co3O4 nanofibers for sensitive and selective glucose detection. Biosens. Bioelectron. 2010, 26 (2), 542−548. (b) Hu, M.; Tian, J.; Lu, H. T.; Weng, L. X.; Wang, L. H. H2O2-sensitive quantum dots for the label-free detection of glucose. Talanta 2010, 82 (3), 997−1002. (c) Jiang, Y.; Zhao, H.; Lin, Y.; Zhu, N.; Ma, Y.; Mao, L. D




(12) Hall, E. A. H.; Gooding, J. J.; Hall, C. E. Redox enzyme linked electrochemical sensors: Theory meets practice. Microchim. Acta 1995, 121 (1), 119−145.

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Glucose Determination in Beverages Using Carbon Nanotube

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