Chemical Education Today
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Research Advances by Angela G. King
Designing Dendrimers To Offer Micelle-type Nanocontainers The class of macromolecules known as dendrimers gets its name from the words for tree (dendron) and part (meros) due to their molecular structure. Dendrimers are comprised of molecular chains that branch out from a common center in a tree-like or generational fashion. The globular shape of high molecular weight dendrimers, coupled with convergent synthetic methodology with the ability to introduce functionally diverse sequences, gives dendrimers the potential to serve as molecular mimics. Researchers in Massachusetts have designed, synthesized, and studied the properties of a dendrimer with hydrophobic and hydrophilic substituents on an orthogonal plane. The resulting polymer contains one of the substituents in its concave interior and the other at the convex surface. This design promotes micelle-like behavior in polar solvent (with the polar substituents on the surface) and inverted micelle arrangement in non-polar solvent.
Figure 2. A cartoon of the polar and apolar nanocontainers. Reprinted with permission from J. Am. Chem. Soc. 2004, 126, 12636–12637. Copyright 2004 American Chemical Society.
Led by S. “Thai” Thayumanavan, the team of researchers used a carboxylic acid group as the hydrophilic group and a decyl chain as the hydrophobic group. After preparing the biaryl monomer, the dendrimers were formed using iterative procedures. The resulting dendrimers were characterized by size exclusion chromatography, MALDI– R–OH RO OR TOF mass spectrometry, and 1H and 13C 1–3 4–6 NMR. To test the formation of micelles that OH O invert with solvent polarity, the researchR= HO O O OH ers employed dye encapsulation studies. O-n-C10H21 O O-n-C10H21 O n-C10H21-O O The dendrimers were treated with potasO sium hydroxide, which deprotonated the n-C10H21-O HO O O O HO O O O O-n-C10H21 O carboxylic acid groups to afford water O O OH O O-n-C10H21 O soluble polycarboxylates. Reichardt’s dye O O HO O O-n-C10H21 H O-n-C O O (pyridinium-N-phenoxide betaine) was 10 21 O-n-C H HO O 10 21 O O HO added and the color of the resulting soluO tion showed that the dendrimers were se* O O-n-C10H21 HO 1&4 questering the hydrophobic dye molecules within their concave interior in a fashion 2&5 * similar to small molecule surfactants such O OH as sodium lauryl sulfate. Researchers were O O-n-C10H21 O HO n-C10H21-O able to estimate the number of dye molOH O O O-n-C10H21O ecules in the interior of each micelle usO O HO O O O ing the dye’s extinction coefficient. The O O O-n-C10H21 third-generation of a dendrimer held an HO O O O-n-C10H21 O
O
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Figure 1. Structures of dendrons and dendrimers employed in the nanocontainers. Six structures are shown. Structures 1, 2, and 3 have OH groups at the positions shown by *. Structures 4, 5, and 6 have the same three R groups attached to the second moeity in the box. Reprinted with permission from J. Am. Chem. Soc. 2004, 126, 15636–15637. Copyright 2004 American Chemical Society.
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Image: I. Willner
average of 8.5 dye molecules in the micelle’s interior, and even first generation dendrimers were able to sequester dye molecules, although many fewer, as the result of aggregation. The dendrimers were soluble in common organic solvents, and they were dissolved in toluene to check for inverted micelle formation. Proflavine, a hydrophilic dye insoluble in toluene by itself, was added to solutions of the dendrimers in toluene. It is hypothesized that the polar nature of the concave interior and specific acid–base interactions between the dye molecules and carboxylic acid groups of the dendrimer were what resulted in solubilizing the proflavine. Fewer dye molecules were sequestered in the inverted micelle (1.5 for third-generation dendrimers) compared to the micelle formed in water. Dynamic light scattering was employed to determine whether polymer properties are unimolecular or due to aggregation. At 105 M concentrations, unimolecular and aggregated containers were formed by different dendrimers. Ultimately the team hopes to design and produce amphiphilic dendrimers with diverse functionality in the particle’s center. When these particles are water soluble, the first step toward structurally complex biomimetic molecules will be achieved.
Figure 3. Biocatalytic AuNPs growth in the presence of glucose.
More Information 1. Vutukuri, D.; Basu, S.; Thayumanavan S. Dendrimers with Both Polar and Apolar Nanocontainer Characteristics. J. Am. Chem. Soc. 2004, 126, 15636–15637. 2. Marino, F. Use of Tangle Links To Show Globular Protein Structure. J. Chem. Educ. 1994, 71, 741. 3. More information on dendrimers can be found in this Journal. Simanek, Eric E.; Gonzalez, Sergio O. Dendrimers: Branching Out of Polymer Chemistry. J. Chem. Educ. 2002, 79, 1222.
Optical Biosensor for Glucose Possible through Biocatalytic Nanoparticle Growth Gold nanoparticles (AuNPs) have many biological applications: they are used as “weight labels” and optical labels for biorecognition events such as DNA hybridization, they can be conjugated to biomaterials to amplify biomaterial binding at the surface, and they catalyze the reduction of metal ions in many biosensors. Such reductions enlarge the nanoparticles as the reduced metal is deposited. Enzymatic enlargement of metallic NPs has been carried out with NAD(P)H cofactors but has remained largely uncharted territory. Now a team of scientists at The Hebrew University of Jerusalem has carried out the catalytic enlargement of AuNPs with the aid of glucose oxidase. This work has been applied to the development of an optical biosensor for glucose that may serve as a model for future work. Absorbance spectroscopy demonstrates the immediate growth of gold nanoparticles when hydrogen peroxide is added to a buffered solution of AuCl4, AuNP seeds, and the surfactant cetyltrimethylammonium chloride. Since both the peroxide and AuNP seeds are critical for the growth, rewww.JCE.DivCHED.org
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Figure 4. Absorbance spectra of solutions containing AuNP seeds, 3 10 10 M, 2 10 4 M HAuCl 4 , 47 g mL 1 GOx, in 0.01 M phosphate buffer solution and CTAC, 2 103 M, upon reaction for 10 min at 30 °C, with different concentrations of -D(+) glucose: (a) 0 M; (b) 2 106 M; (c) 1 105 M; (d) 2 105 M; (e) 5 105 M; (f) 1.1 104 M; (g) 2.4 104 M. Reprinted with permission from Nano Letters 2005, 5, 21–25. Copyright © 2005 American Chemical Society.
searchers postulate that the seeds act as catalysts for reduction of the AuCl4 ions by H2O2 according to the equation − AuCl4 + 3 2 H2O2
AuNP
− + Au(s) + 4Cl + 3H + 3 2 O2
HR–SEM measurements and the particles’ absorbance spectra both indicate that particles increased in size from 12 1 nm to 18 1 nm. This growth is in agreement with
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Reports from Other Journals the value calculated by the Mie theory, an advanced mathematical formula for computing the amount of light scattering by spherical particles. Hydrogen peroxide is generated by many oxidases as they catalyze the oxidation of their substrate with O2. Willner’s team applied their understanding of how AuNP growth was tied to peroxide in designing a new biosensor. If a system contained an oxidase and all components necessary for AuNP growth except H2O2, then addition of substrate and its subsequent enzymatic oxidation would trigger a sudden NP growth as the enzyme catalyzed production of H2O2. Researchers chose glucose oxidation by glucose oxidase (GOx) as their model study. The absorbance spectra of systems (under O2) of AuCl4, CTAC, AuNP seeds, and GOx were monitored over time as different concentrations of glucose were added. As predicted, the absorbance values of NPs increase as glucose concentration increases, presumably due to an increase in peroxide production which enhances AuNP growth if glucose, GOx, or O2 is omitted. No growth in NP size is observed (indicated by no change in the absorbance spectrum), because lack of these molecules would shut down peroxide production. The same method was employed with AuNP seeds bound to an aminopropylsiloxane film. A similar result was obtained and the glass support models detected glucose at a concentration of 2 106 M. Researchers now hope to extend their work to other enzyme systems that produce peroxide. This would allow the detection of additional substrates, such as cholesterol, lactate, and choline.
More Information 1. Zayats, M.; Baron, R.; Popov, I.; Willner, I. Biocatalytic Growth of Au Nanoparticles: From Mechanistic Aspects to Biosensor Design. Nano Letters 2005, 5, 21–25. 2. “Exploring the Nanoworld” is a Web site designed to introduce teachers and students to nano-architectural wonders. It may be found online at http://mrsec.wisc.edu/edetc/index.html (accessed Mar 2005). 3. “Integrating Nanotechnology into the K12 Classroom” is available online at http://www.bowlesphysics.com/nano/ (accessed Mar 2005). 4. JCE Classroom Activity #62 has students explore size-dependent properties of gold nanoparticles. McFarland, A.; Haynes, C.; Mirkin, C.; Van Duyne, R.; Godwin, H. Color My Nanoworld. J. Chem. Educ. 2004, 81, 544A–544B. 5. JCE Classroom Activity #72, published in this issue, has students explore nanofabrication techniques while creating patterns. See Haynes, C.; McFarland, A.; Van Duyne, R.; Godwin, H. Nanopatterning with Lithography. J. Chem. Educ. 2005, 82, 768A–768B.
Carbon Nanotubes and Human Cells? The recent literature is filled with exciting reports on the potential of nanomaterials in engineering and biomaterials. The small size of nanoparticles (1–100 nm) results in increased conductivity and reactivity in comparison to larger particles, and these properties might lead to unique biomedical applications such as drug delivery agents. But ei668
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ther widespread use in engineering fields or a biological application may result in human exposure through inhalation, oral, or dermal pathways, and the realistic potential of nanoparticles is thus dependent on their impact on human health. Single-walled carbon nanotubes that were chemically altered to be water soluble have been shown to enter fibroblasts, T cells, and HL60 cells. Previous work demonstrated that unmodified carbon nanotubes were more toxic than quartz dust or carbon black, two known pulmonary toxicants, if they reached lung tissue. Human exposure to carbon fibers also results in dermal irritation. Immortalized cell lines may be grown indefinitely if the medium is replenished and the culture does not become contaminated. Nanoparticles have been shown to adversely affect immortalized HaCaT human keratinocyte cultures, indicating that they may enter cells. But only very recently did scientists study the interaction of nanoparticles with living human cells. An interdisciplinary team from North Carolina State University led by Nancy Monteiro-Riviere employed high-resolution transmission electron microscopy (HRTEM) and release of proinflammatory cytokine interleukin 8 (IL-8) as a biological response marker to study unmodified multi-walled carbon nanotubes (MWCNT). Researchers used a microwave plasma-enhanced chemical vapor deposition system to grow vertically aligned multiwalled carbon nanotubes (MWCNT) on an 80-mm iron film catalyst. Scanning electron microscopy showed the aligned nanotube film grew perpendicular to the substrate, was approximately 50 m thick, and had a density of 1 1010 nanotubes/cm3. HRTEM images show that the nanotubes have a multi-walled structure that resembles bamboo shoots. The nanotubes were manually removed from the film by scraping. Human neonatal epidermal keratinocytes (HEK) were cultured and exposed to MWCNT in growth medium. MWCNT were suspended in the growth medium by sonification, which had previously been shown not to break or deform the nanotubes. The HEKs were then treated with the MWCNT-laden growth medium for different times before being harvested, fixed, and embedded in agar. Cell monolayers were obtained by growing cells on 60-mm Permanox dishes and treating them with either a solution of 0.4 mg/ mL nanotubes or the same solution filtered to remove aggregated particles. Both the agar-embedded cells and the cell monolayers were embedded in resin prior to TEM analysis. The results clearly show that chemically unmodified MWCNT were present within HEKS and that cells exposed to MWCNT had morphology different from the control. The nanotubes, up to 3.6 mm long, were primarily found in the intracytoplasmic vacuoles of the keratinocytes. While the nuclei of treated cells did not contain MWCNT, the MWCNT were at times found in the free cytoplasm close to the nuclei, perhaps piercing the nuclear membrane. The number of MWCNT found within cells increased as the treatment concentration and exposure time increased. The nanotubes clearly affected the cells they interacted with. HEK viability slightly decreased in a dose-dependent
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Figure 5. Arrows indicate MWCNT present within (a) the cytoplasmic vacuoles of a HEK and (b) intracytoplasm of a Keratinocyte monolayer. HRTEM (c) revealing the bamboo-like structure of MWCNT. Reprinted from Monteiro-Riviere, N.: Namanich, R.; Inman, A.; Wang, Y.; Riviere, J. Multi-walled carbon nanotube interactions with human epidermal keratinocytes. Toxicology Lett., 2005, 155, 377–384. Used with permission of Elsevier.
manner after treatment with nanotubes. Also, IL-8 concentration in the growth media of treated cells was significantly higher than in the control studies. This response may be the result of MWCNT attaching to the plasma membrane and/ or entering the cells. These results clearly indicate that exposure to MWCNT must be evaluated before the risk from exposure in an occupational or environmental scenario can be assessed. An even greater task for scientists is to elucidate the mechanism of penetration and effect.
More Information 1. Monteiro-Riviere, N.; Namanich, R.; Inman, A.; Wang, Y.; Riviere, J. Multi-walled carbon nanotube interactions with human epidermal keratinocytes. Toxicology Lett. 2005, 155, 377–384. 2. This research was highlighted in the December 24, 2004 issue of Science (2004, 306, 2164). Nanotechnology: Ingesting Nanotubes was selected as the editor’s choice, which highlights
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the most recent literature that has major impact in science and medicine. 3. A JCE experiment has students producing carbon nanotubes. Fahland, B. Chemical Vapor Deposition of Carbon Nanotubes: An Experiment in Materials Chemistry. J. Chem. Educ. 2002, 79, 203–206. 4. “Exploring the Nanoworld” is a Web site designed to introduce teachers and students to nano-architectural wonders. Found online at http://mrsec.wisc.edu/edetc/index.html (accessed Mar 2005). 5. “Integrating Nanotechnology into the K–12 Classroom” is available online at http://www.bowlesphysics.com/nano/ (accessed Mar 2005).
Angela G. King is Senior Lecturer in Chemistry at Wake Forest University, P.O. Box 7486, Winston-Salem, NC 27109;
[email protected].
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