In the Laboratory
Using the Enzymatic Growth of Nanoparticles To Create a Biosensor An Undergraduate Quantitative Analysis Experiment James Bai,† Kaleyhia Flowers,†† Salil Benegal, Milagros Calizo, Vrudhdhi Patel, and Sandra Whaley Bishnoi* Department of Biological, Chemical, and Physical Sciences, Illinois Institute of Technology, Chicago, IL 60616; *
[email protected] Nanoparticles have a great range of possible applications in optics and medicine (1–3). Gold nanoparticles, in particular, have size-dependent optical properties owing to the presence of localized surface plasmons. In gold nanoparticles, plasmons are excited through absorption of visible light leading to oscillations of the conduction electrons, which results in a ruby red color and an absorbance of green light (~520 nm) (4). Gold nanoparticles have been used to optically detect and monitor various biological events through the biocatalytic enlargement of the metal nanoparticles (5–7). This nanoparticle enlargement is reflected in the visible spectrum of the solution by a color shift and an increase in the optical extinction (scattering and absorption) of the solution. Enzymatic systems such as glucose oxidase (5, 8), nitrate reductase (9), and alkaline phosphatase (10) have been successfully used to grow metal nanoparticles for the detection of analytes. Glucose oxidase (GOx), for instance, catalyzes the oxidation of β-d-glucose to d-glucono-1,5-lactone. In the process, molecular oxygen is reduced to hydrogen peroxide (H2O2). The H2O2 produced in the GOx reaction then acts as a reductant in the presence of chloroauric acid (HAuCl4), reducing Au3+(aq) to Au0(s), which deposits on the surface of gold “seed” particles in solution. This change in particle size leads to an increase in the optical extinction of the particles in solution arising from a combination of the absorption and scattering from the particles in solution. The glucose oxidase–gold reaction can be conducted at variable concentrations of glucose in an undergraduate laboratory under experimental settings mimicking an optical glucose biosensor. The experiment introduces undergraduate students to applications of nanotechnology while utilizing appropriate instrumental analysis techniques present in an analytical chemistry curriculum (11). Experiment The experimental setup is divided into two parts. In the first part the students verify that the reaction between hydrogen peroxide and chloroauric acid in the presence of gold nanoparticles causes a quantitative response in the gold solution, which is monitored using UV–vis spectroscopy. The second part simulates a glucose biosensor. The students use the enzyme glucose oxidase to produce peroxide in situ, which reduces the Au(III) in solution causing an enlargement of the colloidal gold nanoparticles. †Current address: Department of Bioengineering, University of Rochester, Rochester, NY 14627. ††Current address: College of Arts and Sciences, Chicago State University, Chicago, IL 60628.
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Part I: Reaction of Au(III) with H2O2 Stock solutions of gold colloid, 8 mM H 2 O 2 , and 0.01% HAuCl4 are required for this experiment. In addition, a 0.22 mg/mL solution of bovine serum albumin (BSA) in 0.01 M phosphate buffer (PB) are prepared. The protein–particle interaction with BSA mimics that between the enzyme and the gold surface used in Part II leading to reduced particle aggregation and a larger dynamic range. The experiment will work with commercially available gold colloid (Sigma, St. Louis, MO) or freshly prepared gold colloid solutions (12). Gold colloid can be synthesized using the Turkevich method (12). Briefly, 1 mL of 29 mM HAuCl4 is added to 100 mL of ultrapure water (Millipore, 18 MΩ cm) and brought to a boil, then 1.5 mL of 34 mM sodium citrate is added, and the solution continued to boil for 10 minutes. The nanoparticle growth experiment has been tested with seed particles ranging in size from 5 to 100 nm with similar sensitivities; therefore maintaining an exact particle size is unimportant to conduct the experiment. A 4.5 mL disposable cuvette (1 cm path length) with square cap is used as a reaction vessel and for the UV–vis analysis. To each cuvette, 1 mL of gold colloid (~2.3 × 10‒10 M), 0.6 mL ultrapure water, and 0.75 mL of BSA are added. For the control experiment (0 mM H2O2), 29 μL of HAuCl4 and 1 mL ultrapure water are then added to the cuvette. The solution is then mixed thoroughly, allowed to stand for exactly 10 minutes, and analyzed with the UV–vis spectrophotometer from 500 to 700 nm. Alternatively, the extinction at the wavelength of maximum extinction (~540 nm) can be monitored using a single wavelength spectrometer. The measurement of particles upon reduction of the gold(III) ions with H2O2 is conducted using the same conditions, except that a total of 1 mL of H2O2 diluted in ultrapure water to reach a final concentration ranging from 0 to 1.2 mM is added instead of 1 mL of ultrapure water. Because of the need for consistent reaction times, students carefully record the time after addition of H2O2 and ensure that scans are taken after exactly 10 minutes. The average extinction at 540 nm should be recorded for each H2O2 concentration to plot a standard curve for the extinction versus concentration. Part II: Biosensor Simulation Reaction Stock solutions of gold colloid, 8 mM glucose, 0.01 M phosphate buffer at pH 7, and 0.01% HAuCl4 are required for this part of the experiment. A stock solution containing 0.50 mg/mL of GOx in 0.01 M phosphate buffer solution is also prepared. Each cuvette is initially filled with 1 mL of gold colloid, 0.6 mL ultrapure water, and 0.75 mL of the GOx solution. For the standard curve, six samples are prepared with a
Journal of Chemical Education • Vol. 86 No. 6 June 2009 • www.JCE.DivCHED.org • © Division of Chemical Education
In the Laboratory [glucose]/(mmol L∙1): [H2O2]/(mmol L∙1):
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H2O2 Concentration / (mmol/L) Figure 1. (A) Extinction spectra of gold colloid/Au(III) solutions after exposure to various additions of H2O2 (final concentrations). Extinction increases due to reduction of gold ions onto the gold nanoparticle “seeds” present in solution. (B) Change in the extinction of the gold colloid/Au(III) solution at 540 nm as a function of H2O2 final concentration. The response is linear from 0–0.75 mM H2O2 and saturates at higher concentrations.
Figure 2. Particle enlargement through the reaction of glucose and glucose oxidase: (A) UV–vis spectra of gold nanoparticle solutions after exposure to various concentrations of glucose (0–2.12 mM) and (B) change in extinction at 540 nm versus final glucose concentration demonstrating the linear response between 0 and 1.4 mM glucose. Error bars represent the standard deviation from three measurements.
final glucose concentration ranging from 0 to 2 mM. These samples are generated via careful addition of varying volumes of an 8 mM stock solution of glucose and ultrapure water to make a total volume of 1 mL. After addition of the glucose solution and 29 μL of HAuCl4 to the cuvette, the cuvettes are sealed and mixed thoroughly to allow complete reaction. As before, the UV–vis spectrum are recorded after a 10 minute incubation period. From the average spectra at each concentration, the extinctions at 540 nm are obtained to plot a standard curve for the extinction versus final concentration of glucose.
Results
Hazards Chloroauric acid is a highly corrosive acid that causes severe burns upon contact with the skin, eyes, the respiratory tract, mouth, and stomach. Chloroauric acid should be kept away from heat and light for extended periods. Hydrogen peroxide is corrosive and hazardous even when diluted and can cause burns upon contact with the skin, eyes, mouth, and respiratory tract. Goggles and gloves should be used at all times when handling these solutions.
Reaction of Au(III) with H2O2 Addition of H2O2 reduces Au(III) ions in solution and plates elemental gold onto the nanoparticles, increasing the extinction of the solution (Figure 1A). The nanoparticle enlargement causes a change in the color and clarity of the reaction mixture. The initial light-red or purple reaction mixture changes to a darker color as the reaction progresses. This is reflected in the spectra taken at each H2O2 concentration level: the peak wavelengths red shift and the extinction increases. The average change in extinction is found by recording the average extinction at 540 nm for each H2O2 concentration and subtracting the average extinction recorded at 0 mM H2O2. From the plot of the change in extinction at 540 nm versus the concentration of the peroxide solution (Figure 1B), a linear relationship is observed. The system approaches saturation at a peroxide concentration of 1.0 mM. The linear portion of the data provides the calibration for the change in particle extinction as a function of H2O2 concentration. The linear least-squares
© Division of Chemical Education • www.JCE.DivCHED.org • Vol. 86 No. 6 June 2009 • Journal of Chemical Education
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In the Laboratory
regression gives a R2 value of 0.99, indicating high linear correlation in the system. This standard curve can be used to measure the concentration of H2O2 in an unknown sample. Biosensor Simulation Reaction Glucose oxidase reacts with glucose to produce d-glucono1,5-lactone and H2O2. As the peroxide is produced, it reduces the gold ions in chloroauric acid to elemental gold that is deposited on the colloid particles. The nanoparticle enlargement is measured by the increasing extinction in the UV–vis spectra with varying concentrations of glucose reacting with glucose oxidase (Figure 2). The average change in extinction is found by recording the average extinction at 540 nm for each glucose concentration and subtracting the average extinction recorded at 0 mM glucose. The sensitivity of the gold colloid system with glucose and glucose oxidase is linear between concentrations of 0 and 1.3 mM glucose (Figure 2A). A standard curve plotting average change in extinction at 540 nm against glucose concentration shows the linearity of this system (Figure 2B) and can be used as a calibration curve to determine the levels of glucose in an unknown sample. The R2 value in the calibration curve is 0.965, indicating linear correlation between the glucose concentration in the system and the extinction at 540 nm. Conclusion This experiment provides students with the opportunity to create a hydrogen peroxide sensor though gold nanoparticle enlargement. The experiment serves to expose students to the sizedependent properties of gold nanoparticle extinction, as well as the concept of biosensor design. The growth of nanoparticles can be linked to the enzymatic reaction between glucose oxidase and glucose, which can be used to monitor biological processes. Using UV–vis spectroscopy we have demonstrated that the resulting reaction provides quantitative results at lower peroxide and glucose concentrations, but saturates at higher concentrations. This laboratory experiment design provides students with an example of how nanoparticle-based biosensors function and demonstrates the difference between linear and dynamic ranges. Acknowledgments We thank the National Science Foundation for support of K.F. and J.B. (Research Experience for Undergraduates, National Science Foundation Grant Number 0552896). Materials support for this work was provided by the Department of Bio-
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logical, Chemical and Physical Sciences at Illinois Institute of Technology. The authors wish to thank Y. J. Lin and Y. Huang for assistance with UV–vis measurements and the reviewers for careful reading of the text. Literature Cited 1. Fortina, P.; Kricka, L. J.; Graves, D. J.; Park, J.; Hyslop, T.; Tam, F.; Halas, N. J.; Surrey, S.; Waldman, S. A. Trends in Biotechnology 2007, 25, 145–152. 2. Michalet, X.; Pinaud, F. F.; Bentolila, L. A.; Tsay, J. M.; Doose, S.; Li, J. J.; Sundaresan, G.; Wu, A. M.; Gambhir, S. S.; Weiss, S. Science 2005, 305, 538–544. 3. Weissleder, R. Nat. Biotech. 2001, 19, 316–317. 4. Colloidal Gold: Principles, Methods, and Applications; Hayat, M. A., Ed.; Academic Press: San Diego, CA, 1989. 5. Willner, I.; Baron, R.; Willner, B. Adv. Mater. 2006, 18, 1109– 1120. 6. Bharde, A.; Kulkarni, A.; Rao, M.; Prabhune, A.; Sastry, M. J. Nanosci. Nanotechno. 2007, 7, 4369–4377. 7. Shang, L.; Chen, H.; Deng, L.; Dong, S. Biosens. Bioelectron. 2008, 23, 1180–1184. 8. Willner, I.; Baron, R.; Willner, B. Biosens. Bioelectron. 2007, 22, 1841–1852. 9. Kumar, S. A.; Abyaneh, M. K.; Gosavi, S. W.; Kulkarni, S. K.; Pasricha, R.; Ahmad, A.; Khan, M. I. Biotechnol. Lett. 2007, 29, 439–445. 10. Basnar, B.; Weizmann, Y.; Cheglakov, Z.; Willner, I. Adv. Mater. 2006, 18, 713–718. 11. For additional experiments involving nanoparticles see Njagi, J.; Warner, J.; Andreescu, S. J. Chem Educ. 2007, 84, 1180–1182. Oliver-Hoyo, M.; Gerber, R. W. J. Chem Educ. 2007, 84, 1174–1176. Solomon, S.; Bahadori, M.; Jeyarajasingam, A. V.; Rutkowsky, S. A.; Boritz, C.; Mulfinger, L. J. Chem Educ. 2007, 84, 322–325. Dungey, K. E.; Muller, D. P.; Gunter, T. J. Chem Educ. 2005, 82, 769–770. 12. Turkevich, J.; Stevenson, P. C.; Hillier, J. J. Phys. Chem. 1953, 57, 670–673.
Supporting JCE Online Material
http://www.jce.divched.org/Journal/Issues/2009/Jun/abs712.html Abstract and keywords Full text (PDF) Supplement Instructions for the students
Journal of Chemical Education • Vol. 86 No. 6 June 2009 • www.JCE.DivCHED.org • © Division of Chemical Education
