Uptake and Impact of Silver Nanoparticles on Brassica rapa: An

Laboratory Experiment pubs.acs.org/jchemeduc

Uptake and Impact of Silver Nanoparticles on Brassica rapa: An Environmental Nanoscience Laboratory Sequence for a Nonmajors Course Kevin M. Metz,* Stephanie E. Sanders, Anna K. Miller, and Katelyn R. French Department of Chemistry, Albion College, Albion, Michigan 49224, United States S Supporting Information *

ABSTRACT: Nanoscience is one of the fast growing fields in science and engineering. Curricular materials ranging from laboratory experiments to entire courses have been developed for undergraduate science majors. However, little material has been developed for the nonmajor students. Here we present a semester-long laboratory sequence developed for a nonmajors course, where students investigate the potential environmental impacts of nanoscience. Students synthesize and characterize silver nanoparticles using green synthetic methods. They then use the suspension of silver nanoparticles to “water” Wisconsin Fast Plants, Brassica rapa, over a three to four week period to simulate environmental exposure. Possible impacts are examined throughout the growth period, and silver uptake by the plants is quantified at the end of the growth period. This lab requires design input from the student, making it an open-ended experiment. Although designed for nonmajors, this lab could easily be adapted for an environmental chemistry or chemical nanoscience course. KEYWORDS: First-Year Undergraduate/General, Upper-Division Undergraduate, Environmental Chemistry, Laboratory Instruction, Hands-On Learning/Manipulatives, Nonmajor Courses, Nanotechnology, Plant Chemistry, UV-Vis Spectroscopy become practitioners in their chosen field. When educating nonmajors, the learning goals shift from specific content to broad understanding of science. That is to say, a nonmajors course should improve scientific literacy, increase the understanding of the scientific method through laboratory exploration, and help students make connections between science and their chosen field of study. The laboratory sequence described here is a semester long, open-ended, that is, explorative, experiment that was implemented in a nanoscience course taught in Albion College’s Prentiss M. Brown Honors Program. Students in the honors program are required to take four discussion-based seminar courses, one in each division of the College. Many honors students who are not majoring in STEM disciplines use the seminar in Natural Sciences and Mathematics to fulfill the College’s laboratory course requirement. As a result, the seminar on nanoscience was populated by upper-level students who have actively avoided science coursework and is thus considered a nonmajors course. Nanoscience was selected as the theme for the course because it is state-of-the-art in science and its cross-disciplinary nature facilitated student interests and engagement, which helped keep the students excited to learn. This laboratory sequence supported the class exploration of nanoscience through the synthesis, characterization, and exploration of a single nanomaterial, silver nanoparticles.

N

anoscience, or the study of matter with at least one dimension between 1 and 100 nm that displays properties different from its bulk counterpart,1 is a rapidly growing, cross-disciplinary field. The perceived importance of nanoscale research to fundamental science can be illustrated by federal funding through the National Nanotechnology Initiative, which has grown from $464 million in 2004 to $1.85 billion in 2011.1 One can also consider the number of consumer goods that self-report the inclusion of nanomaterials, which has risen from 54 in 2005 to 1317 in 2010,2 as a predictor of the economic importance of nanoscience through commercialization. In hopes of preparing our undergraduate students for graduate studies or careers in science, many instructors have incorporated concepts of nanoscience into their courses,3−7 while others have created entire coursed based on the subject.8−11 Several laboratory experiences have been published in support of such efforts, offering undergraduate students hands-on experience in synthesizing and characterizing nanomaterials.12−19 One area of curricular development that is currently lacking, however, is laboratory experiments directed toward students majoring in areas outside of science, technology, engineering, or mathematics (STEM) disciplines, referred to here as nonmajors. The learning goals for a nonmajors course are inherently different from the learning goals for a course designed for STEM students. When educating STEM students, the overarching goals often are to teach enough content knowledge and provide enough hands-on experience for the students to © 2013 American Chemical Society and Division of Chemical Education, Inc.

Published: December 13, 2013 264

dx.doi.org/10.1021/ed400177r | J. Chem. Educ. 2014, 91, 264−268

Journal of Chemical Education

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Laboratory Experiment

LABORATORY SEQUENCE In this semester-long laboratory experience, students synthesize aqueous solutions of silver nanoparticles (AgNPs), characterize the AgNPs by ultraviolet−visible spectroscopy (UV−vis), add varying quantities of the aqueous suspensions to Wisconsin Fast Plants20 at regular intervals for a specified amount of time, and then analyze the plants for silver uptake by inductively coupled plasma optical emission spectroscopy (ICP-OES). The laboratory section of the course met weekly for 3 h each week. There were 11 students in the course, who worked as individuals throughout the lab. The data presented here is compiled, student-generated data used to illustrate the overall laboratory sequence. The weekly activities of this semester-long laboratory sequence are outlined in Table 1. The first week of lab is

needed to determine the correct dilution factor for the reductant. Each student, or pair, explored at least 2 natural reductants for the synthesis of silver nanoparticles. Characterization of the synthesized AgNPs was performed using UV−vis spectroscopy. Students were instructed on the use of the instrument and they collected their own spectra. UV−vis spectra were collected as quickly as 15−30 min after the reductant was added to the solution. Multiple spectra were collected, up to a week later, to confirm the end of nanoparticle growth and to examine nanoparticle stability. Representative student UV−vis data for AgNPs synthesized with Maxwell House instant coffee and a 1:100 dilution of grape juice are shown in Figure 1. Both spectra display a characteristic plasmon

Table 1. The Schedule of Weekly Activities Utilized in the Described Experiment Week

Topic

1 2 3 4 5 6 7 8 9 10 11 12 13 14

Laboratory Safety and Orientation Measurements and Observations Synthesis of AgNPs Characterization of AgNPs Measurement and Water Fast Plants Measurement and Water Fast Plants Measurement and Water Fast Plants Measurement and Water Fast Plants Dehydration of Plants Digestion of Plants Characterization of Digested Plants Data Analysis and Interpretation Presentations Clean-up and Check-out

Figure 1. Representative UV−vis spectra for silver nanoparticles synthesized with Maxwell House instant coffee and a 1:100 dilution of store-bought grape juice.

dedicated to a laboratory tour, safety instruction, and proper use of a notebook. The second week of the lab students are introduced to basic glassware through a short exercise (see the Supporting Information). The third week of the semester marks the start of the students’ investigation of silver nanoparticles, which provides time to discuss the basics of nanoscience in the discussion or lecture portion of the course. All subsequent weeks are devoted to carrying out the exploration of the interaction between silver nanoparticles and Brassica rapa.

peak that can be used to determine the AgNP diameter.14 A list of reductants used, the plasmon peak wavelength (λmax) measured by UV−vis spectroscopy, and their calculated size are displayed in Table 2. Sizes were calculated by assuming a Table 2. The Natural Reductant and Resulting UV−Vis Peak Absorbance and Calculated Silver Nanoparticle Diameter

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Reductant Maxwell House Instant Coffee Maxwell House Instant Decaffeinated Coffee Black Tea Green Tea Honey Pomegranate Juice (1:100 dilution) Grape Juice (1:100 dilution) Red Wine Red Wine Vinegar

SYNTHESIZING AND CHARACTERIZING SILVER NANOPARTICLES To synthesize AgNPs, students followed previously published protocols.21−25 Several different synthetic strategies were discussed in the course and the students selected to use natural products as the reductants (please see Supporting Information for more details on the selection process). In this approach, students used tea, coffee, fruit juice, wine, or honey as the reductant to synthesize nanoparticles (NPs). By allowing the students to select their reductant, they became more engaged in this sequence and reported to have a sense of ownership. For example, many students chose to use their own teas or coffee, rather than those provided in the laboratory. To perform the synthesis, students prepared a 0.1 M stock solution of AgNO3, and then mixed 1 mL of this stock solution with 6 mL of their selected reductant solution at room temperature. In some cases, for example, fruit juice, some experimentation was

λmax/nm

Calculated NP Diameter/nm

450 475

54 72

445 450 460 460 430 470 480

51 54 61 62 40 68 75

linear relationship between particle size and the absorbance peak maximum. A correlation plot was developed by extracting λmax values from data published by Sigma-Aldrich Chemical Co. for AgNPs of known size (see the Supporting Information).26 The correlation equation, which had a R2 value of 0.984, was λmax = 1.425 × diameter (nm) + 372.56 265




Figure 2. (A) Mean height of plants exposed to 100 μL of AgNP solution, synthesized with caffeinated coffee, once a week or twice a week and plants exposed only to water, as a function of days after planting. (B) For comparison, the mean height of plants exposed to water, coffee solution, and 0.1 M Ag(NO3)(aq) (100 μL twice a week) as a function of days after planting. Differences in the traces are not statistically different. All plants were exposed to the same total volume of liquid per week, with a twice per week exposure. Water was used to compensate in experiments were the spike was delivered only once per week.

population. This is most likely because the acidity of the coffee remaining in the AgNP solution lowered the pH of the soil to an inhospitably range for the plant growth. Plants were terminated between 18 and 28 days of growth, when pollination is required and seedpod production occurs. Pollination was intentionally not performed in this study. At this point the entire plant was removed from the soil, washed with copious amounts of deionized water, and dried in an oven at 110 °C for at least one week. The dried plants were combined by category, weighed, pulverized, and then digested in a small volume of concentrated nitric acid (5−10 mL depending on the volume of plant mass). The acidic solution was then filtered through a 0.45 μm filter, diluted to a known volume in a volumetric flask (typically 25 mL) and analyzed by using ICP-OES or ICP-MS. Students were instructed on the operation of the ICP-OES and collected their own data. Verification of low concentration was completed by the instructor using ICP-MS. The range of silver concentrated in the dried plant biomass depending on the volume and frequency of exposure is shown in Table 3. Table 3 contains data for AgNPs solutions made with Maxwell House coffees because this was the only reductant used by multiple groups with different dosing conditions. The silver content ranged from 14 to 2200 ppb depending on the volume of AgNPs suspension delivered and the frequency of delivery. The other reductants resulted in silver concentrations within this range with similar results based on volume and frequency. Separation between roots and stems−leaves revealed that about two-thirds of the silver was in the roots and the rest was in the stems−leaves. The presence of silver in the stems−leaves clearly indicates uptake of silver by the plants, while the remaining detected silver may result, in part, from nanoparticle adsorption to the roots. An interesting result is the concentration of silver found in plants that were exposed to ionic silver solutions. These plants had silver concentrations that were one thousand-fold greater than plants exposed to AgNP solutions. Overall, it is unclear from the collected results if the plants were taking up silver nanoparticles or residual ionic silver. There was insufficient

This equation yielded similar results to previously published sizing data.14 The AgNP solutions were stable, that is, neither any change in UV−vis spectra nor flocculation was observed, over the entire length of the experiment.

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INVESTIGATING PLANT GROWTH IN THE PRESENCE OF SILVER NANOPARTICLES Once AgNPs were synthesized and characterized, students used the AgNP suspensions to “water” Wisconsin Fast Plants, Brassica rapa,20 over a three to four week period to simulate environmental exposure. Wisconsin Fast Plants were sowed and grown according to the directions included in the plant kits, purchased from Carolina Science Supply. Silver nanoparticle suspensions, which contained the reductant, i.e., coffee, tea, etc., were added to the top of each compartment using micropipets. Students decided what volumes to add and how often to add them. Typical volumes ranged from 100 to 1000 μL, added 1 to 3 times per week. By allowing the students to determine the exposure schedule, a greater sense of ownership was achieved, as well as a better understanding of the challenges presented by designing an experiment to mimic environmental events in a laboratory setting. To monitor the possible impact of AgNPs on plant growth, the heights of the plants were measured every few days. Figure 2A shows the average height of plants that were exposed to 100 μL of AgNP suspension once and twice a week (red squares and blue triangles, respectively) compared to a water control (black dots). Figure 2B shows the average height of plants that were exposed to a coffee solution in the absence of AgNPs (green diamonds), a 0.1 M solution of AgNO3 (purple stars), and the same water control data (black dots). There are no statistical differences in height between the AgNP exposed plants and the control plants (error bars are omitted because their overlap obstructs the data). Thus, it can be observed exposure to coffee, ionic silver, or AgNP solutions has no negative impact on plant growth under the experimental conditions. It should be noted, however, students who exposed their plants to 1000 μL of AgNPs suspensions (1 to 3 times per week) experienced plant losses exceeding 60% of the 266




Students reported that they learned mimicking an environmental process in a manner that can be monitored in the lab is surprisingly challenging. Students were also surprised to learn the importance of control groups and the need for standard methods to produce comparable results. Overall, students felt they had a better understanding of how “science is done” and felt they had a greater ability to critically analyze scientific results by the end of this sequence. These should be two learning goals of any nonmajors science course.

Table 3. The Concentration of Silver Measured per Mass of Dried Plant Biomass for Different Reductants and Plant Dosing

Reductant Maxwell House Instant Coffee Maxwell House Instant Decaffeinated Coffee Maxwell House Instant Coffee Maxwell House Instant Decaffeinated Coffee Maxwell House Instant Coffee Maxwell House Instant Decaffeinated Coffee Ionic Silver Control (no reductant) a

Volume Delivered/μL

Frequency/ per week

Total Number of NP Spikes

1000

3a

12

2200

1000

3a

12

1500

100

2b

6

35

100

2b

6

25

100

1b

3

22

100

1b

3

14

100

2b

6

20300

Concentration of Silver in Dried Biomass (ppb)

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SUMMARY An open-ended semester-long laboratory sequence was developed for the purpose of teaching nonmajors the scientific process in a manner that mimics research in the chemical sciences as closely as possible. Students had significant input in the design of the experiments and were thus able to compare their results with those of their peers to understand how small changes in methodology affect experimental outcomes, resulting in an appreciation for controls, statistical analysis, and standard methods. Although the described laboratory sequence was specifically developed for nonmajors, it is easily adaptable for an environmental chemistry or nanochemistry course. It could be used as a central theme for such a class given the interest in the uptake and toxicity of nanoparticles by plants,27,28 and the general concern about the impact of nanomaterials on the environment.29−31

Dosed over a 4 week period. bDosed over a 3 week period.

time during the semester to attempt to quantitatively determine the ionic silver concentration present in the AgNP solutions. However, this result, while uncertain, presents an important learning opportunity for students as it calls for discussions on experimental design and the need for additional experiments. These discussions can be used to highlight challenges of scientific exploration, such as the quest for absolute certainty based on a statistical approach. It should be noted that silver concentrations were below the detection limit in the water and coffee solution control plant groups.

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ASSOCIATED CONTENT

S Supporting Information *

Instructions for the students; notes for the instructor; student survey. This material is available via the Internet at http://pubs. acs.org.

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AUTHOR INFORMATION

Corresponding Author

HAZARDS Students should wear goggles and follow general laboratory safety precautions at all time while in the laboratory. Silver nitrate is a corrosive material that can cause burns on contact with skin and eyes. The use of gloves while handling silver nitrate is recommended. Waste containers must be available for any waste that contains silver or silver nanoparticles. The toxicity of AgNPs in unclear at this time, thus gloves and careful handling are encouraged when working with the AgNP suspensions. Concentrated nitric acid is caustic and should only be worked with in a fume hood.

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Acknowledgment is made to the Donors of the American Chemical Society Petroleum Research Fund for partial support of this research, and to David Carey, Kurt Hellman, and the Albion College Department of Biology for assistant with Fast Plants.

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STUDENT LEARNING Student learning in this laboratory was assessed using a simple survey administered at the beginning and the end of the course (see the Supporting Information). Students were asked to rank their understanding of nanoscience, the scientific method, and scientific experimental design using a Likert scale. The terminal survey also contained an open-ended question asking students to identify what knowledge they gained that surprised them. The majority of students reported “I know nothing about this topic” with respect to nanoscience or experimental design at the beginning of the course. Slightly over half of the class reported having “I have a basic understanding of this topic” or “I am familiar with this topic” regarding the scientific method at the beginning of the course. By the end of the course, all of the students reported “I am familiar with this topic” of “I am knowledgeable with this topic” with respect to all three fields.

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Uptake and Impact of Silver Nanoparticles on Brassica rapa: An

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