Preliminary Observations of the Role of Material Morphology on

Aug 16, 2010 - Protein-Electrophoretic Transport in Gold Nanocomposite Hydrogels ..... the transport of proteins through PAM gels at low electrical fi...
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Preliminary Observations of the Role of Material Morphology on Protein-Electrophoretic Transport in Gold Nanocomposite Hydrogels Jeffery W. Thompson, Holly A. Stretz,* and Pedro E. Arce Department of Chemical Engineering, Tennessee Technological UniVersity, CookeVille, Tennessee 38505

Nanocomposite polymeric hydrogels have potential to play an important role in clinical diagnostics, therapeutic agents, and electroanalytical devices, among other biotechnological applications. However, the relationship between nanocomposite structure (morphology) and transport specifically of proteins has not been systematically described. In this study, polyacrylamide (PAM) nanocomposites have been synthesized containing various compositions and aspect ratios of gold nanoparticles (GNP). These nanocomposite hydrogels have been characterized for morphology, and examined for their ability to change the effective electrophoretic mobility of a model protein, ovum serum albumin (OSA), under a low applied electric field of 6.7 V/cm. Addition of spherical (low aspect ratio) gold nanoparticles reduces the effective mobility of OSA, a result that cannot be explained by the lower effective cross-link density noted in swelling studies. However, the effective mobility of OSA can be predicted using simple tortuous path models, specifically the Lape-Cussler. An increase in aspect ratio of the nanoparticles produced further reductions in mobility, and this reduction was so significant that tortuous path contribution could not explain it. We expect that percolation of the higher aspect ratio gold nanoparticles (as seen in TEM images) led to preferred conduction through the gold network, and therefore resulted in lower mobility in the buffer. The structure-mobility relationships found here help establish one possible regime for transport of proteins through nanocomposite hydrogels. 1. Introduction Polymeric hydrogels play an important role in clinical diagnostics as a separation medium for biomolecules. Some difficulties for electrophoretic separations of proteins in hydrogels persist however. For example, common separations may use sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), in which the sample requires postpurification in order to analyze the permeate by mass spectroscopy.1 Non-native changes in the protein structure may not be restored during this “purification” step, therefore any improvements in PAGE which leave the protein in its native form and still obtain separation would be very beneficial. Further, polyacrylamide gels (PAM) must often be manipulated by hand during staining, etc., but the mechanical properties and shelf life of the PAM are typically very poor.2 Nanocomposite hydrogels offer significantly improved mechanical properties over traditional nonreinforced hydrogels, including improvements in strength and elongation,3 making these novel materials excellent candidates for nextgeneration diagnostics. For example, responsive nanocomposite gels (formed from poly(N-isopropylacrylamide) or PNIPAM and montmorillonite) offer a variety of properties including increased elongation at break, improved optical clarity at high temperatures,4 higher effective cross-link densities,5 greater uptake,6 faster response time, and even enhanced conductivity.7 Nanocomposite hydrogels have potential application as therapeutic agents,8,9 and electroanalytical diagnostic devices10 in addition to diagnostic separations. However, very little information is available about how proteins transport through these nanocomposite hydrogels during electrophoresis.11,12 A significant body of work exists describing how DNA moves through various hydrogels based on first principles, and Stellwagen has given an excellent review of this work,13 however proteins (most likely) will transport differently in hydrogels due to the globular * To whom correspondence should be addressed. Tel.: 931-372-3495. Fax: 931-372-6352. E-mail: [email protected].

conformation under native PAGE conditions versus rod or hookshaped geometries commonly assumed for describing DNA motion. Regarding the current work on proteins in nanocomposite gels, Huang et al. showed how hydrophilic multiwall carbon nanotubes (MWNT) can be used in polyacrylamide hydrogels to achieve the otherwise difficult separation of apolipoprotein A-I and complement C3 under native PAGE conditions. Yu et al. have shown how using gold nanoparticles in conjunction with capillary electrophoresis can facilitate separation of acidic and basic proteins, though the role of filler morphology was not described. As noted by Fu et al., filler morphology plays an important role in protein separations for a single channel nanofluidic device.14 Electrophoretic separation of proteins is believed to depend on both molecular sieving15 and differential mobility due to charge density differences in the analytes. Since the channel structure is known to affect separation of proteins in a nanofluidic device,16,17 then nanoparticle shape and geometrical properties such as the separation distance between impermeable nanoparticles and the channel morphology should also play roles in the capacity for a nanocomposite hydrogel to perform protein separations. This paper outlines the possible effect of nanoparticle shape, specifically nanoparticle aspect ratio on protein transport under an electrophoretic driving force at low electrical fields. These conditions were chosen to study the potential role of the tortuous path effect for molecular sieving of these macromolecules. The nanoparticle of choice in this study was gold for three reasons: (1) gold has a low areal charge density on its surface and therefore charge-based/electrical double layer interactions with the analyte will be minimized, (2) gold nanorods are available in a variety of aspect ratios, and (3) gold nanoparticles are expected to be nearly immobilized in the gel throughout electrophoretic testing.10,18 Future efforts will focus on the more complicated effects expected at the higher electrical fields typical of clinical protein separations.

10.1021/ie100291b  2010 American Chemical Society Published on Web 08/16/2010

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2. Experimental Section 2.1. Materials. Gold chloride trihydrate, and N,N′-methylenebis-acrylamide (BIS) were obtained from Sigma-Alrich. Silver nitrate, sodium chloride, L-ascorbic acid, HPLC grade water, acrylamide, ammonium persulfate (APS), tetramethylethylenediamine (TEMED), and sodium hydroxide were obtained from Fisher Scientific. Sodium borohydride was obtained from Alfa Aesar. Cetyltrimethylammonium bromide (CTAB), and trisborate EDTA (TBE) buffer were obtained from Ameresco. Citrate-stabilized 5-nm gold colloid (5 × 1013 particles/mL) was obtained from Ted Pella. Monodisperse gold nanorods coated in CTAB produced via a seed-mediated synthesis19 were obtained from Dr. Catherine J. Murphy’s group at University of Illinois at Urbana-Champaign. Albumin from egg white (MW ) 45 000) (OSA) was obtained from Acros Organics. All materials were of the highest purity available and were used as received. 2.2. Surfactant-Assisted Gold Nanoparticle Synthesis (SA-GNP). Gold nanoparticles were produced in gram-scale quantity using methods described in the literature.20 This literature method was selected because high quantities of gold nanorods could be produced. Briefly, 3.67 g of CTAB and 0.85 g of AgNO3 were sonicated with 50 mL of HAuCl4 (50 mM) for 10 min. The gold was reduced to Au3+ with 10 mL of 1 M ascorbic acid, noted by a color change from orange to white, and subsequently, nanoparticles were formed upon complete reduction with 1 mL of 0 °C NaBH4 (0.5M), noted by a color change from white to dark purple. Digital image analysis of TEM images was performed to characterize the average aspect ratio. 2.3. Seed-Mediated Gold Nanoparticle Synthesis (SMGNP). Gold nanoparticles were prepared using a seed-mediated procedure.21 This method was selected because it has been shown to achieve gold nanoparticle aspect ratios as high as 25. Three vials were labeled A, B, and C. Solutions A and B contained HAuCl4 (2.2 mM), NaOH (2.5 mM), CTAB(0.1 M), and ascorbic acid (10 mM). Solution C contained HAuCl4 (1.1 mM), NaOH (1 mM), CTAB(0.1 M), and ascorbic acid (5 mM). One milliliter of the 5-nm gold colloid (Ted Pella) was added to solution A (the seed). One milliliter of A was transferred to solution B, and then all of B was quickly transferred to solution C where nanoparticle growth continued overnight. Upon completion of reaction nanoparticles were dark brown. Digital image analysis of TEM images was performed to characterize the average aspect ratio. 2.4. Templated Gold Nanoparticle Synthesis (T-GNP). Gold nanotubules were fabricated using template wetting nanofabrication described previously by Steinhart et al. This method was selected because of the ability to produce aspect ratios of greater than 50. These materials were provided thanks to Katherine Hudson, Steven Bearden, and Dr. Scott Gold at Louisiana State University. A wetting solution of 15 µL of 1 wt.% chloroauric acid (HAuCl4, ACS Reagent, Sigma Aldrich) in acetone was applied to a porous alumina membrane with nominal pore diameter of 200 nm (Whatman Anodisc) which served as the template. The precursor was reduced to gold metal and annealed in air for 2 h at 200 °C followed by 1 h at 350 °C. Any excess gold was removed from the exterior of the porous alumina template by reactive ion etching in helium (30 min, 200 mtorr, 25 W). Nanotubules were freed from the template by immersing the gold-coated templates in 0.5 M aqueous KOH overnight while stirring slowly. The nanotubules were then centrifuged, decanted, and dispersed in 0.5 M aqueous KOH twice to ensure the removal of the alumina. Once the

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alumina was removed, the process was repeated with ten cycles in water to replace the 0.5 M aqueous KOH solution. The gold nanotubules were manually dispersed and stored in water. After receipt at Tennesse Technological University (TTU) they were redispersed using 200 µL of HNO3/mL gold nanotubule solution received. 2.5. Composite Hydrogel Synthesis. SA-GNP were recovered via centrifugation to remove free dissolved CTAB. The precipitate was resuspended in HPLC-grade water and centrifugation was repeated until no foam appeared on shaking. Stock hydrogel solutions containing acrylamide (0.82 M), N,N′-methylene-bis-acrylamide (12.9 mM), and various compositions of Au nanoparticles (SA-GNP volume fraction, ΦAu ) 0.0087-0.055; SM-GNP volume fraction, ΦAu ) 0.0087-0.02; T-GNP volume fraction, Φ ) 0.006; and monodisperse gold nanorods volume fraction, ΦAu ) (2.52-25.2) × 10-7) were prepared via sonication of the components in 60 mL of HPLC grade water for a period of at least 3 h. Ten mL of this solution was initiated with 50 µL of APS (0.44M) and 5 µL of TEMED. The gels were then allowed to polymerize at room temperature between two glass plates with a comb insert for a period of at least 3 h prior to use. 2.6. Gel Electrophoresis. The gels were immersed in a TBE buffer, pH ) 8.0. Dansyl chloride labeled albumin (OSA), at 40 µL of 1 mg/mL concentration, was loaded into the gel lanes. Gel electrophoresis was performed at constant voltage, 6.67 V/cm, for a period of 45 min using a Fisher FB1000 power supply. (Note the current was not specifically controlled.) After performing electrophoresis, the gels were placed under a UV lamp to measure the protein band position, and then imaged using a digital camera and copy stand apparatus. 2.7. TEM Analysis. TEM images for surfactant-assisted and seed-mediated gold nanorods were produced by Dr. Jibao He at Tulane University. Samples were prepared at TTU on carboncoated 200 mesh copper grids. Images were taken using a JEOL 2011 High-Resolution TEM at 40 µPa vacuum. These images were analyzed manually using Adobe Photoshop by Julie Shell at TTU. The average aspect ratio reported is a number average. Between 50 and 250 particles were analyzed. TEM images for monodisperse gold nanorods were taken at University of IllinoisUrbana. These images were analyzed manually with particle count 528. 2.8. Swelling Experiments. Cast hydrogels two inches thick were first cut into rectangular blocks. These blocks were subjected to 100% acetone for a period of 2 h to remove any excess water. The length, width, and thickness of the gel were measured using digital calipers to obtain total gel volume V ) L × W × T. The blocks were subsequently immersed into 100 mL of HPLC grade water and placed in individual Ziploc bags in a temperature controller at 60 °C. Block measurements were then taken using the procedure above on samples retrieved from the liquid and patted dry at various time steps. 3. Results and Discussion 3.1. TEM Nanoparticle Analysis. In Figure 1, a representative TEM image of the SA-GNP low aspect ratio gold nanoparticles is shown. Figure 2 presents the frequency analysis for all of the nanoparticles imaged. The distribution is skewed, and the average aspect ratio was determined to be 1.3 ( 0.6. This corresponds to particles that are nearly spherical in shape. These nanoparticles are between 10 and 20 nm in diameter. In Figure 3 a representative TEM image of the SM-GNP sample is shown. These gold nanoparticles were polydisperse with an

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Figure 1. TEM image of low aspect ratio surfactant-assisted gold nanoparticles (SA-GNP).

Figure 3. TEM image of seed-mediated gold nanoparticles (SM-GNP).

Figure 4. Histogram of aspect ratio distribution for SM-GNP obtained via TEM analysis.

Figure 2. Histogram of aspect ratio distribution for SA-GNP obtained via TEM analysis.

average aspect ratio determined to be 2.9 ( 2.8, as shown in Figure 4, the frequency analysis results. The short dimension was approximately 40-100 nm; and the long dimension varied from 45 nm to 2 µm. Figure 5 presents a representative ESEM image of the T-GNP sample. ESEM analysis was performed at TTU in the Center for Manufacturing Research. Figure 6 presents a representative TEM image of the monodisperse nanorods, and Figure 7 gives the frequency analysis results. The short dimension was determined to be approximately 20 nm; and the long dimension was approximately 80 nm. The average aspect ratio was determined to be 3.9 ( 0.9. 3.2. TEM Nanocomposite Hydrogel Analysis. Nanocomposite hydrogels were formed by depositing 2 µL of the gel solution on the surface of a TEM grid at 2 different compositions represented by Figures 8 and 9. In Figure 8, nanoparticles appear

to form discrete clusters or islands within the hydrogel. At higher loadings, shown in Figure 9, the nanoparticle domains begin to overlap creating chains or networks. Please note that the unstained polymer appears as the white background. 3.3. Swelling. To further characterize the properties of the composites, the equilibrium swelling information of hydrogel composites was compared to swelling for gels containing only organic cross-linkers. In Figures 10 and 11, swelling data for multiple compositions of the PAM/SA-GNP gels and the PAM gels with BIS cross-linker are compared. The hyperbolic function fit shown is a guide for the eye. For organically crosslinked hydrogels, increasing the composition of BIS caused the equilibrium swelling to decrease. By contrast, as the volume of gold nanoparticles in the composite increased at constant crosslinker concentration (% C, defined as (wBis)/(wBis + wPAM)), the swelling of the gel increased, with the greatest swelling extent noted at ΦAu ) 0.0261. (Note that in both cases four samples were tested, though data for only two representative cases are shown here for the purposes of clarity.)

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Figure 7. Histogram of aspect ratio distribution for monodisperse gold nanorods obtained via TEM analysis.

Figure 5. ESEM image of high aspect ratio templated gold nanoparticle sample (T-GNP).

Figure 6. TEM image of monodisperse gold nanorods.

Figure 8. TEM image of polyacrylamide/surfactant-assisted gold nanoparticle, SA-GNP-based nanocomposite as polymerized on grid ΦAu ) .0087.

Extent of swelling of a hydrogel (S) scales with cross-link density, νe, as νe-0.48 (see Pacios et al.22). This is consistent with our data for organically cross-linked gels. Regarding the nanocomposites, Haraguchi et al. have seen a decrease in swelling on introduction of montmorillonite filler particles to PNIPAM hydrogels, and they have attributed this to crosslinking of PNIPAM with montmorillonite.4 They, for instance, noted a shift in the lower critical solution temperature, an indicator of chemical bonding between the PNIPAM and the montmorillonite surface, which is not possible for the gold-PAM system. The increase in swelling noted for our composites may therefore be due to interference with cross-linking by the filler particle. Nevertheless, changes in swelling indicate that the morphology of the gold nanocomposite hydrogels has been altered by the presence of the filler. 3.4. 1-D Gel Electrophoresis. Vertical gel electrophoresis was performed on nanocomposite hydrogels to obtain an

effective mobility of a model protein, OSA. Table 1 contains a complete listing of experimental parameters. A low electrical field (driving force) was selected for this study, 6.7 V/cm, to minimize the expected effects of the electro-osmotic contribution. In Figure 12, the normalized electrophoretic mobility is presented, here defined as the mobility of the OSA in the nanocomposite gel divided by the mobility of the OSA in a control gel at the same cross-linker content. For all electrophoresis experiments, the organic cross-linker content was kept at 3% C. For the SA-GNP-based nanocomposites the effective mobility was reduced as filler composition in the composite increased. For gels with higher aspect ratio SM-GNPs, the effective OSA mobilities were lower than what would be achievable using the SA-GNPs and simply increasing volume fraction. Thus protein movement could be altered more by the aspect ratio of the particle than by the composite composition. For gels with template-synthesized gold nanoparticles (with an

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Figure 11. Swelling of gold composite hydrogels at different compositions at 60 °C normalized by the volume of the gel at zero hours is shown. Lines are present to guide the eye.

Figure 9. TEM image of polyacrylamide/surfactant assisted gold nanoparticle, SA-GNP-based nanocomposite as polymerized on grid ΦAu ) .06.

Figure 10. Swelling of hydrogels of different compositions at 60 °C normalized by the volume of the gel at zero hours is shown. Lines are present to guide the eye.

even higher aspect ratio) mobilities again were lower than achievable by the highest composition of the previous sample. (Only one data point is shown due to the limited availability of this synthesized material.) For gels with monodisperse gold nanorods mobilities were lower than SA-GNP or SM-GNP, but in the same range as seen for T-GNP (see Figure 12a). While a direct comparison between the monodisperse gold nanorod data and the other nanoparticle-based data is difficult to achieve due to the great differences in volume fraction (a practical constraint arising from expense of the highly monodisperse materials) the monodisperse gold nanorod data may be fit using an exponential function as shown in Figure 12a and then comparison of the projection with other data shown in Figure 12b. This extrapolation places the volume fractions on the same scale. In Figure 12b the monodisperse gold nanorods were shown to produce mobilities which are far less than that of the spherical gold nanoparticles.

Huang et al. reported a different result, that protein mobilities increased for Triton X assisted nanoTiO2 and nanoAl2O3 in PAM.12 Furthermore, Huang et al. showed a mobility increase for gels containing Triton-X coated carbon nanotubes. In both cases the effect of aspect ratio is difficult to determine because of the ability of Triton X to solubilize proteins. In summary, the native PAGE data presented here demonstrate that nanofiller aspect ratio has a substantial effect on protein mobility, reducing the transport of proteins through PAM gels at low electrical fields. All values in Figure 12b are the result of multiple tests averaged, and the error bars are included to show reproducibility. The error bars are not detectable on the plot scale for some data points, though all error bars are plotted. 3.5. Tortuosity Arguments. Models such as those presented by Nielson23 for monodisperse particles, and Lape-Cussler24 for polydisperse particles, have been used for predicting the relative permeability of composite membranes for gaseous diffusion. The hydrogels used in this research could be viewed as an approximation of polymer “sieving” media, except that the driving force is electrical- instead of pressure-based. Thus membrane permeability models might offer some insight into the behavior of the composite hydrogel system. These models are superimposed upon the low aspect ratio SA-GNP-composite data in Figure 13. The model of Nielson, in this case, overpredicts the effective mobility of the protein. The LapeCussler model seems to fit for the range of SA-GNP compositions tested. Practical constraints, including difficulty with crosslinking and visual clarity of the gel, limit use of GNPs at higher compositions, so that testing this hypothesis over the entire volume fraction range is not practical. However, for the range tested, a tortuosity argument based upon polydisperse particles inserted into the hydrogel seems to be useful to predict the effective mobility. In Figure 12b, the SA-GNP-based composite data along with the other nanocomposites is presented to show the effect of aspect ratio. For SM-GNP-based composites (recall that these are slightly higher aspect ratio) the tortuosity model does not predict the experimental reduction in mobility. The protein mobilities found using templated gold nanoparticle-based composites were not predictable using the Lape-Cussler tortuosity model. The error in such a prediction was even higher than that for the seed-mediated gold nanoparticle-based composites. Finally, Figure 12b shows that the monodisperse gold nanorods appear to be the least consistent upon extrapolation. Another surface area dependent factor affecting the motion of the proteins inside the nanocomposite gel may be electro-

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Table 1. Measured Current as Function of Filler Aspect Ratio GNP

AR

composition

voltage (V/cm)

time (min)

current (mA)

SA-GNP SM-GNP T-GNP monodisperse GNP monodisperse GNP

1.3 3.0 (polydisperse) ∼50 3.9 3.9

0.0087-.055 0.0087-.02 0.006 (2.52-5.04) × 10-7 (7.56-25.2) × 10-7

6.67 6.67 6.67 6.67 6.67

45 45 45 45 45

1 2 10 1 2

osmosis; Matos et al., for example, have reported increases in electro-osmotic flow for silica-based nanocomposite hydrogels.25 In Matos’ work, the silica inclusions had a local surface charge, but the particles were in fact insulators. By comparison, the gold nanoparticles in the present work can conduct on a bulk scale if the composite is percolated. We have some evidence that the high aspect ratio particles produced a percolated morphology, as seen in the TEM images in Figure 9. Thus a second explanation is possible for the reduced protein mobilites seen in the gold nanogels. We postulate that much of the current for the higher aspect ratio filled gels was transmitted through the networked filler. Under this assumption, the current would not be transmitted through the buffer solution where it would have directly contributed to the transport of the charged proteins. In fact, this effect could be measured, as shown in Table 1. The overall current increased with higher aspect ratio nanoparticles. Unsurprisingly, gold conducts. A similar result, higher conductivity gels, has been reported for platinum-based agarose composites,26 but the effect of aspect ratio was not determined in that system. Further, it should be noted that the Pt-agarose

Figure 13. Tortuous path models of Lape-Cussler and Nielson versus SAGNP-based nanocomposite experimental mobilities for OSA protein.

composites enhanced mobility rather than reducing mobility which, again, should originate with the surface charge of the platinum nanoparticle in relation to the analyte particle. In summary, the higher aspect ratio could have led to percolation and short circuiting. However, it remains of fundamental importance that at low aspect ratio and low electrical field, tortuosity arguments are useful in predicting protein transport properties such as effective mobility in hydrogels as shown in this work. Other experimental conditions would need further efforts to understand and predict the behavior of protein transport inside the nanocomposite gels. 4. Summary and Concluding Remarks

Figure 12. (a) Effect of aspect ratio on electrophoretic mobility of OSA. Mobility is normalized by dividing the mobility of OSA in a nanocomposite by the mobility of OSA in a polyacrylamide control gel. • ) SA-GNP composites. 2 ) SM-GNP composites. 9 ) T-GNP composites. · · · ) Monodisperse gold nanorod composites (extrapolated). (b) Normalized OSA mobility using monodisperse gold nanorod composites.

Polyacrylamide nanocomposites have been synthesized containing various compositions and aspect ratios of gold nanoparticles. These nanocomposite hydrogels have been characterized for morphology, and examined for their ability to change the effective electrophoretic mobility of a model protein, ovum serum albumin (OSA), under an applied electric field of low voltage. Furthermore, at low electric fields, the hypothesis was that a tortuous path created by high aspect ratio nanoparticles would influence the transport of the protein. In terms of morphological characterization, the spherical gold nanoparticles appear to have formed well dispersed clusters, while the higher aspect ratio materials (as determined by TEM) appear to have formed percolated networks or nanochains. Equilibrium swelling behavior of the nanocomposite hydrogel acted in a manner consistent with lower effective cross-link density, and this effect was greater on a volume percent basis than addition of BIS cross-linker. Regarding transport of the protein under electrophoresis, addition of gold nanoparticles reduces the effective mobility of OSA, a result that cannot be explained by the effective cross-link density. Moreover, the effective mobility of this complex charged molecule in the nanocomposite with essentially spherical nanoparticles can be predicted using simple tortuous path models, specifically the Lape-Cussler model which fit better than a Nielsen model. However, an increase in

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aspect ratio of the nanoparticles produced a further reduction in mobility, and this reduction was so significant that tortuous path contribution alone could not explain it. Based on the experimental evidence, we expect that one possible reason for this behavior is that the percolation of the higher aspect ratio gold nanoparticles led to preferred conduction through the gold network, and therefore resulted in lower effective protein mobility in the buffer. The structure-mobility relationships found here help to promote understanding of the regimes associated with the transport of proteins through nanocomposite hydrogels. Further efforts are needed to completely elucidate (and predict) the motion of proteins inside nanocomposite gels. These will be the subject of future contributions. Acknowledgment We thank Dr. Jibao He, Tulane University, for providing TEM images, Julie Shell at TTU for doing the histograms, as well as Katherine Hudson, Steven Bearden, and Dr. Scott Gold at Louisiana State University for providing the templatesynthesized high aspect ratio gold nanoparticles, and Sean Sivapalan, Stefano Boulos, and Dr. Catherine J. Murphy at University of Illinois at Urbana-Champaign for providing monodisperse gold nanorods. J.T. gratefully acknowledges being a recipient of a University Diversity Fellowship from Tennessee Technological University. We are indebted to the continued support from the Center for Manufacturing Research of Tennessee Technological University. Literature Cited (1) Sinha, S.; Kosalai, K.; Arora, S.; Namane, A.; Sharma, P.; Gaikwad, A. N.; Brodin, P.; Cole, S. T. Immunogenic Membrane-Associated Proteins of Mycobacterium tuberculosis Revealed by Proteomics. Microbiology 2005, 151, 2411–2419. (2) Okay, O.; Wilhelm, O. Polyacrylamide-Clay Nancomposite Hydrogels: Rheological and Light Scattering Characterization. Macromolecules 2007, 40, 3378–3387. (3) Haraguchi, K.; Farnworth, R.; Ohbayashi, A.; Takehisa, T. Compositional Effects on Mechanical Properties of Nanocomposite Hydrogels Composed of Poly(N, N-dimethylacrylamide) and Clay. Macromolecules 2003, 36 (15), 5732–5741. (4) Haraguchi, K.; Takehisa, T.; Simon, F. Effect of Clay Content on the Properties of Nanocomposite Hydrogels Composed of Poly(N-isopropylacrylamide) and Clay. Macromolecules 2002, 35 (27), 10162–10171. (5) Haraguchi, K. Nanocomposite Gels: New Advanced Functional Soft Materials. Macromol. Symp. 2007, 256, 120–130. (6) Churochkina, N. A.; Starodoubtsev, S. G.; Khokhlov, A. R. Swelling and Collapse of the Gel Composites Based on Neutral and Slightly Charged Poly(acrylamide) Gels Containing Na-Montmorillonite. Polym. Gels Networks 1998, 6, 205–215. (7) Zhao, X.; Ding, X.; Deng, Z.; Zheng, Z.; Peng, Y.; Long, X. Thermoswitchable Electronic Properties of a Gold Nanoparticle/Hydrogel Composite. Macromol. Rapid Commun. 2005, 26, 1784–1787. (8) Wijaya, A.; Hamad-Schifferli, K. Ligand Customization and DNA Functionalization of Gold Nanorods via Round-Trip Phase Transfer Ligand exchange. Langmuir 2008, 24 (18), 9966–9969.

(9) Owens, D. E., III; Eby, J. K.; Jian, Y.; Peppas, N. A. Temperatureresponsive polymer-gold nanocomposites as intelligent therapeutic systems. J. Biomed. Mater. Res., Part A 2007, 83A (3), 692–695. (10) Kazimierska, E. A.; Ciszkowska, M. Thermoresponsive Poly-Nisopropylacrylamide Gels Modified with Colloidal Gold Nanoparticles for Electroanalytical Applications. 1. Preparation and Characterization. Electroanalysis 2005, 17 (15-16), 1384–1395. (11) Yu, C.-J.; Su, C.-L.; Tseng, W.-L. Separation of Acidic and Basic Proteins by Nanoparticle-Filled Capillary Electrophoresis. Anal. Chem. 2006, 78 (23), 8004–8010. (12) Huang, G.; Zhang, Y.; Ouyang, J.; Baeyens, W. R. G.; Delanghe, J. R. Application of carbon nanotube-matrix assistant native polyacrylamide gel electrophoresis to the separation of apolipoprotein A-I and Compliment C3. Anal. Chim. Acta 2006, (557), 137–145. (13) Stellwagen, N. C.; Stellwagen, E. Effect of the Matrix on DNA Electrophoretic Mobility. J. Chromatogr., A 2009, 1216, 1917–1929. (14) Fu, J.; Schoch, R. B.; Stevens, A. L.; Tannenbaum, S. R.; Han, J. A patterned anisotropic nanofluidic sieving structure for continuous-flow separation of DNA and proteins. Nat. Nanotechnol. 2007, 2, 121–128. (15) Cox, H. C.; Teven, J. M. G. On the Mechanism of the MolecularSieve Effect in Polyacrylamide Gel Electrophoresis. J. Chromatogr., A 1976, 123 (2), 261–270. (16) Fu, J.; Mao, P.; Han, J. Artificial Molecular Sieves and Filters: A New Paradigm for Biomolecule Separation. Trends Biotechnol. 2008, 26 (6), 311–320. (17) Zeng, Y.; Harrison, D. J. Self-Assembled Colloidal Arrays as ThreeDimensional Nanofluidic Sieves for Separation of Biomolecules on Microchips. Anal. Chem. 2007, 79, 2289–2295. (18) Grimm, A.; Nowak, C.; Hoffman, J.; Schartl, W. Electrophoretic Mobility of Gold Nanoparticles in Thermoresponsive Hydrogels. Macromolecules 2009, 42, 6231–6238. (19) Murphy, C. J.; Sau, T. K.; Gole, A. M.; Orenndorf, C. J.; Gao, J.; Gao, L.; Hunyadi, S. E.; Li, T. Anisotropic Metal Nanoparticles: Synthesis, Assembly, and Optical Applications. J. Phys. Chem. B 2005, 109, 13857– 13870. (20) Jana, N. R. Gram-Scale Synthesis of Soluble, Near-Monodisperse Gold Nanorods and Other Anisotropic Nanoparticles. Small 2005, 1 (89), 875–882. (21) Busbee, B. D.; Obare, S. O.; Murphy, C. J. An Improved Synthesis of High-Aspect Ratio Gold Nanorods. AdV. Mater. 2003, 15 (5), 414–416. (22) Pacios, I. E.; Molina, M. J.; Gomez-Anton, M. R.; Pierola, I. F. Correlation of Swelling and Crosslinking Density with the Composition of the Reacting Mixture Employed in Radical Crosslinking. J. Appl. Polym. Sci. 2007, 103, 263–269. (23) Nielsen, L. E. Models for the Permeability of Filled Polymer Systems. J. Macromol. Sci., Part A: Pure Appl. Chem. 1967, 1, (5), 929– 942. (24) Lape, N. K.; Nuxoll, E. E.; Cussler, E. L. Polydisperse Flakes in Barrier Films. J. Membr. Sci. 2004, 236, 29–37. (25) Matos, M. A.; White, L. R.; Tilton, R. D. Electroosmotically Enhanced Mass Transfer Through Polyacrylamide Gels. J. Colloid Interface Sci. 2006, 300, 429–436. (26) Bhattacharya, S.; Chanda, N.; Liu, Y.-S.; Grant, S. A.; Gangopadhyay, K.; Sharp, P. R.; Bashir, R.; Gangopadhyay, S. Enhanced DNA Separation Rates in Nano-Platinum Doped Agarose. J. Bionanosci. 2008, 2, 1–8.

ReceiVed for reView February 5, 2010 ReVised manuscript receiVed July 21, 2010 Accepted July 26, 2010 IE100291B