Role of Nanocomposite Hydrogel Morphology in ... - ACS Publications

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Ind. Eng. Chem. Res. 2010, 49, 11866–11877

Role of Nanocomposite Hydrogel Morphology in the Electrophoretic Separation of Biomolecules: A Review Jyothirmai J. Simhadri,† Holly A. Stretz,*,† Mario Oyanader,†,‡ and Pedro E. Arce*,† Department of Chemical Engineering, Tennessee Technological UniVersity (TTU), CookeVille, Tennessee 38505, and Department of Chemical Engineering, UniVersidad Catolica del Norte, Antofagasta, Chile

Hydrogels are widely used to produce biomolecular separations in electrophoretic applications, where gel morphology and charge effects combine to produce the separation. Nanocomposite gels are poised to revolutionize this field in both improved handling characteristics and improved separations. Gel morphology and charge effects have traditionally been manipulated by varying the copolymer composition, but recent reports show that novel morphological changes can also be induced in the gel using templating methods and nanoparticle addition. To aid realization of the potential for novel electrically driven separations arising from such novel morphologies, we review here advances in materials development alongside a review of the directions in biomolecule/hydrogel electrophoretic transport modeling. Models for polyelectrolyte transport that are potentially useful for understanding novel behaviors caused by gel morphology are analyzed first. With the perspective of theoretical guidance, we then survey nanocomposite hydrogel morphologies keyed by the nanoparticle and matrix. Finally, we survey as well reports of dramatic improvements in the mechanical properties that may be the key to the early adoption of these new materials in biomolecular separation applications. For modeling, we have identified the different scales involved in biomolecular transport in these materials and provided a taxonomy of transport models that emphasize the role of gel morphology in determining both polyelectrolyte mobility and diffusion. Three aspects for future work unique to nanocomposite gel materials are described: (a) descriptions of the distribution of nanoparticles within the gels; (b) descriptions of the motion of the buffer solution that may include electroosmotic effects for nanoparticles with surface charges; (c) descriptions of the motion of the polyelectrolyte molecule inside the new gel material for a given application. These aspects will help to uncover further quantitative details about the ability of these gels to be tunable for differential mass transport of a given type of molecule. Standard gels, currently, lack a broad flexibility to increase the separation of biomolecules and, in general, are not tunable. 1. Introduction Gels are widely used as the separation media for biomolecules such as DNA and proteins in electrophoretic applications.1 Within the past decade, novel gel materials with a modified internal morphology have been synthesized and are available for a wide range of applications including drug delivery, separation of biomolecules with relevance in clinical diagnostics, electrokinetic pumping, and sensor applications, among others. In this contribution, within the framework of the gel morphology, and although we will mention other approaches to modify it, we will mostly cover nanocomposite gels, i.e., gels with nanoparticles embedded in the matrix. The presence of these colloidal particles can bring a significant change to the chemical physics of the gel morphology, in addition to expanding the multiscale nature of the material. In Figure 1, various scales relevant to transport in these materials are depicted, and the addition of nanoparticles to the matrix can affect the performance of the gel on virtually every scale shown. The nanocomposite hydrogel performance can differ from that of the pure component at the macroscopic scale in terms of opacity and cross-link heterogeneity. At the media scale, the nanocomposite can act to improve the gel strength and elongation. At the microchannel scale, individual nanoparticles can potentially interact with the analyte by forming channel walls whose localized charge and geometry will be different from those of * To whom correspondence should be addressed. E-mail: parce@ tntech.edu (P.E.A.), [email protected] (H.A.S.). † Tennessee Technological University. ‡ Universidad Catolica del Norte.

the organic gel. At the nanoscale, the nanoparticles can force transport of the analyte through interparticle channels where flow is now governed by a stronger effect of the electrical double layer because of the small distances between the analyte and channel walls (e.g., a nanochannel). Further, the incorporation of nanoparticles can alter the gel cross-link density or other such direct interactions with the matrix polymer. Because the morphology of traditional gels is known to play a very important

Figure 1. Nanocomposite gels associated with multiscale domains. Nanoparticle inclusion can affect the gel strength, opacity, and matrix heterogeneities on the media and microscopic scales. They can also affect the cross-link density, pore structure, and electrical double layers on the microchannel scales and nanoscales.

10.1021/ie1003762  2010 American Chemical Society Published on Web 08/25/2010

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Table 1. Different Approaches to Modifying the Internal Gel Morphology modification

agent

comment

composition

polyacrylamide (PAM), agarose

templated

DNA, xanthan, sodium dodecyl sulfate, liquid crystals, molecular imprinting

nanocomposite hydrogels

silicate-based, metal nanoparticles, magnetic nanoparticles, microgel particles

anamolous slow mobilities observed for curved DNA molecules in PAM gels that correlate to the acrylamide concentration not the gel pore size selectively alter electrophoretic transport of macromolecules with dimensions approximating those of templating solutes have a wide variety of applications from stimuli-responsive sensors and actuators to microfluidics, pharmaceutical, and biomedical devices

role in the transport of the polyelectrolyte,2-4 nanocomposite gels represent new opportunities for modifying such transport, with implications in separations, barrier and release properties, as well as new opportunities for improving the balance of the mechanical properties. Markets for new technologies based on new materials in separations clearly exist. For example, Candiano et al. have recently described advances in proteome separation technology using soft immobiline gels to address the poor transfer of large proteins trapped in immobilized pHgradient (IPG) strips.5,6 New smart nanomaterials, nanocomposite hydrogels that are mechanically robust or have longer shelf lives, or nanomorphologies that offer improved windows for separation could represent the next frontier of end-user control. The current review focuses on selected types of nanocomposite hydrogels useful in electrically driven separations. We present first some relevant models for polyelectrolyte transport that will aide in connecting new gel morphologies with the potential for novel separations. Next we review reports of morphological changes to the gel arising from interaction with the nanoparticle and affecting its ability to perform primarily as a molecular sieve. Finally we include recently reported improvements in the hydrogel mechanical properties arising from nanoparticle addition. 2. Transport Models Relevant to Gel Morphology Hydrogels are three-dimensional (3-D) networks of chemically or physically cross-linked polymers having a porous structure at the nanometer scale and retaining a high percentage of water. The pore morphology has been modified historically by changing the cross-link density or composition and has been described by a number of authors, reviewed classically by Righetti, and reviewed recently by Stellwagen and Stellwagen.2,7-9 More recently, the pore morphology has been modified by templating and/or by the addition of nanoparticles to be described following a review of theoretical implications of modified pore morphologies. Table 1 summarizes some recent reports and authoritative reviews demonstrating a growing interest in these modified hydrogel morphologies, and Table 2 previews a number of recent contributions demonstrating the variety of nanoparticles under investigation for modification of hydrogels. As mentioned in section 1, we are interested in understanding the role of the morphology of the gel material on enhancing the separation efficiency for polyelectrolyte mixtures in nanocomposite hydrogels. However, only a few isolated publications fully reflect the role that nanoparticles play in determining polyelectrolyte separations in bulk hydrogels. To provide a foundation for further contributions to this important field, we review here current advances for transport models for standard

key ref Stellwagen et al.96,97

Rill et al.54-59 and Byrne and Salian98 Schexnailder and Schmidt99 (see Table 2 also)

gels with special attention to the role of gel morphology. At the end of this section, important modeling advances in nanocomposite gels are mentioned, and finally we describe the directions in which the field is growing. In general and from a macroscopic (or practitioner) point of view, there are two key transport parameters that control the separation efficiency: the mobility and diffusion of the polyelectrolyte within the separation media. The final band position (within gel media, for example) will depend upon the competition between the mobility of the polyelectrolyte and its diffusion. If a favorable situation arises, a reasonable separation of multiple polyelectrolyte bands will occur because of a differential in mobilities combined with appropriate limitations on diffusion. Such transport coefficients depend upon a series of variables and parameters of the system including the magnitude and type of the applied electrical field, the state and type of the polyelectrolyte molecule, and the type of separation media. Figure 2 shows a model taxonomy [this taxonomy acts as a calibrator for the complexity of the models and gives the reader a potential roadmap to guide the development of more accurate and reliable models (see the last section of this review)] based on three key elements needed to model the systems, i.e., electrical field (EF), polyelectrolyte (PE), and separation media (SM). The complexity of the mathematics needed to resolve such models and predict something useful about the system increases as the reader advances outward from the origin in this taxonomy. In this section, we review some of the key models reported in the literature, and related to this taxonomy, to predict the values of such parameters. In particular, we are interested in models useful for understanding the role played by the morphology of the gel media on affecting the values of the transport parameters under a variety of conditions. Table 3 classifies the efforts into four main subfamilies helpful for understanding the role of morphology in determining the values of the polyelectrolyte mobility as well as its diffusion. The first category offers approaches based on “free volume” to predict the values of mobility. The next category, “geometrical domains”, accommodates useful contributions that are based on a geometrical characterization of the separation media. The third category, “scaling arguments”, includes models that describe the motion of the polyelectrolyte molecule within the separation media with respect to certain scales (see Figure 1). Finally, the “direct computational simulation approaches” group includes models based on simulation efforts assisted by a numerical solution approach. We will briefly overview the different aspects of the categories and then refer to several authoritative reviews with additional useful details. A section describing the suggested directions in modeling for nanocomposites specifically is presented last.

silver Fe3O4

P(NIPAM-co-AM)

spherical (120 nm) PNIPAM-co-SA PVA

PAM

spherical (13 nm)

spherical (3 nm) 5 µm

PNIPAM

spherical (5-10 nm)

PAM PAM PNIPAM

spherical (40-250 nm)

carbon nanotube PNIPAM microgels

PNIPAM

PAM

neutral/anionic/cationic PAM PNIPAM

PVA

PDMAM

PAM

PNIPAM

polymer

spherical (50 µm)

preformed clay matrixes

clay aerogel

PNIPAM/PEG-DA microgels gold

spherical (7-15 nm)

organically modified

exfoliated platelets (30 nm diameter, 1 nm thickness)

characteristics of the particle

silica

clay

nanoparticle

Table 2. Characteristics and Potential Applications of Nanocomposite Gels

solvent-switchable electronic properties swelling changes by optical activation add electrical properties localized heating of ferrogel in alternating magnetic fields

structural homogeneity, rapid swelling/deswelling, excellent mechanical strength, LCST behavior excellent resilience, low hysteresis, ultrahigh elongation structural homogeneity, tough mechanical strength with large elongations, large swelling ratios, rapid deswelling responses lower surfactant concentration increases hydrogel network strength, higher amounts make the gel a viscous solution high absorbing character and improved mechanical strength enhanced thermal transition and rapid transition rates modified electrokinetic properties of gel low-density thermoresponsive composite with enhanced mechanical strength changed the mobility of proteins thermosensitive optical properties high equilibrium swelling ratio and faster response rates thermoswitchable electrical conductivity

characteristics of the nanocomposite gel

sensors, catalytic, antibacterial hyperthermia

sensors, photothermal drug delivery devices, actuators, electrochemical, microfluidic valves

biomedical and biotechnological

sensors, controlled delivery devices, thermoresponsive switches bioseparation media controlled release, sensors

biosensors

high shear applications

sensors, actuators, regulators, controlled-release devices, biomaterials

potential application

Mohan et al.84 Lao and Ramanujan87

Sershen et al.62

Pardo-Yissar et al.82

Wang et al.,80 Zhao et al.,81 and Guiney et al.100

Zhang and Chu78

Huang et al.76 Musch et al.77

Bandi et al.64

Matos et al.41 and Hill46

Liang et al.73

Churochkina et al.74

Liu and Hoffmann75

Haraguchi et al.93

Okay and Oppermann72

Haraguchi63

key selected ref

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Figure 2. Taxonomy (basic system components and levels of complexity) in the separation of polyelectrolytes under an applied electric field. The higher the number value in the axis, the more complex the model would be. Table 3. Classification of the Transport Models Relevant to Gel Morphology with Selected Early Contributions type

basic characteristics

free volume geometrical domains (lattices, tubes, channels) scaling arguments

direct computational simulation approaches

early ref

The gel is assumed to act as a sieve with a distribution of pore sizes. The model views the gel as a series of geometrical domains of 1-D, 2-D, or 3-D either single or interconnected. A flexible and thin polyelectrolyte molecule moves inside a small-diameter tunnel where the small length scale dynamics is not important. The molecule moves like a reptation-like style inside the gel. There is direct numerical simulation of the polyelectrolyte motion under an applied electrical field. Polyelectrolyte is modeled by a series of charged beads connected by either springs or freely hinged links. Media are viewed as 2-D or 3-D arrays of obstacles.

2.1. Free Volume. One of the first efforts to predict the mobility of the polyelectrolyte inside a gel was the free-volume model where the gel is assumed to act as a sieving media (with a distribution of pore sizes) and the separation is assumed to be an electrically driven filtration. The model proposed a rather simple relation between the mobility of the polyelectrolyte (inside the gel), µ, and that of the molecule in free solution, µ0, which is given by the fractional volume, f, available to the particle in the gel. Thus, the basic model equation is µ )f µ0

(1)

Now, the fractional free volume, f, may be computed by the simple idea of representing the particle by objects and then estimating the value by using the statistics of the intersection between the particle and the components of the gel distributed as random media of intersecting fibers. Ogston10 was the first to perform the calculation for spherical particles in a suspension of long fibers. Giddings et al.11 proposed to approximate the obstacles by thin sheets to accommodate two-dimensional (2D) objects. The results were generalized to a series of geometries and situations by Chambrach and co-workers.12-14 There is an excellent discussion of the different results for f in section IV

10

Ogston (1958), Giddings et al. (1968),11 and Rodbard and Chrambach (1970)12 Michaels (1958),28 Zimm (1988),101 Zimm (1991),34 Slater and Guo (1995 and 1996),18-20 and Zimm (1996)27 de Gennes (1971),102 Lumpkin and Zimm (1982),103 and Lerman and Frisch (1982)104

Slater and Noolandi (1985),105 Olvera de la Cruz et al. (1986),106 Deutch (1987),107 Deutch and Madden (1989),108 and Shaffer and Olvera de la Cruz (1989)109

(Gel Electrophoresis of Globular Particles) in the review by Viovy15 as well as the relation of models to the empirical equation proposed by Ferguson16 for mobility in gels. Slater et al.17 have offered additional useful comments on the interpretation of eq 1 in terms of the concentration of the gel media and the molecular size of the polyelectrolyte. They also mentioned that, in the limit of the electrical field going to a zero value, the following relationship holds: D µ(E) ) lim Ef0 µ0 D0

(2)

Here D is the diffusivity and E is the electrical field. This relationship suggests (as the authors pointed out) that all results, obtained for thermal diffusion at the zero field limits, can be viewed as low mobility data and vice versa. This observation also suggests the relation of the mobility and diffusion as key transport parameters and the importance of factors affecting them on enhancing the separation. Slater’s group has made substantial contributions to verify the validity of eq 1, and this will be discussed in the Geometrical Domains section. 2.2. Geometrical Domains. In an effort to assess the validity of the prediction of eq 1, Slater’s group18-20 proposed a geometrical-based domain approach to estimate the reduced

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mobility. Thus, they used 2-D square lattices with periodic boundary conditions where obstacles can be placed either periodically or randomly. Therefore, the model allows for the testing of several types of gel morphologies and the role they play in the reduced mobility, µ/µ0. Details about the implementation and solution of this approach have been reviewed extensively either by the authors17,21 or by others15 (section IV.b). Key conclusions are that the model predictions agree reasonably well with those based on the free volume for low fields and low gel concentrations when using compact analyte molecules. When the concentration of gel obstacles increases, the predictions based on the free-volume model are always higher than the ones based on the square-lattice model. In particular, the periodic gels show higher values than those for randomly distributed gels. Slater et al.17 have summarized the comparisons among the different situations, including those for a 3-D lattice22 by using a generalized polynomial function of the obstructed volume Φ, of the reduced mobility, that makes the comparison with the predictions based on the free-volume model very effective: µ ) 1 + a1φ + a2φ2 + a3φ3 + ... µ0

(3)

The conclusion of this comparison is that, for the type of lattice configurations studied, coefficient a1 * 1 and the others aj, j g 2, are always different from zero. This implies that they contradict the values consistent with the model based on the free volume. Slater et al.17 offer an excellent review and discussion on the possible interpretation and limitations of the free-volume model. The results based on Slater’s proposed model for the computation of the reduced mobility are in agreement with other independent analyses.23-25 One important and useful implication of the studies mentioned above is that they show the crucial role played by the morphology of the gel material. They also show the potential role of the dimensions and/or the isotropic or anisotropic nature of the materials.25,26 All of these results are quite useful for further studies of new materials with a modified internal morphology such as the nanocomposite gels of interest to this contribution. Zimm27 used 1-D and 2-D lattice models with periodic boundary conditions to study the effect of the free-energy distribution over the lattice surface on the mobility of the polyelectrolyte. The author’s motivation was to study the role of the interactions of the polyelectrolyte gel and the electrostatic interactions with fixed charges of the gel network. He used a differential type of equation for diffusion, under the presence of a force field, with both a diffusion current contribution and a field-driven current. This equation is solved by using the periodic boundary conditions, and the flux is given in terms of concentrations and potential values at the different nodes of the lattice. The potential features two contributions, i.e., the one associated with the analyte-gel interaction and the other associated with the external electrical field. A value for the field was selected and a Boltzmann distribution chosen for the concentration. The free-energy surface is selected from a Gaussian distribution, and it is given in terms of a “roughness” factor that handles the energy surface characteristics. The analysis for the 2-D lattice requires a numerical solution, but its simplification to 1-D yields an analytical result for the mobility in terms of exponential functions of the free energy and the field values. This result is useful to encapsulate the role of the field value on the mobility for different conditions within the separation media. For example, all of the values of the

mobility predicted by the model reach a plateau at high values of the field. A different plateau is present at low values of the field. There is a distribution of mobility values located between the two plateaus as the roughness factor is varied. Very high values of the roughness factor show the least mobility, and the inverse is also possible. The author offered an analysis of the role of the bumps and valleys as well as assessed the role of the 1-D versus the 2-D lattice models. The gel can be described by a noninteracting 1-D array of tubes, but they overestimate the effect of the roughness of the energy landscape. However, through tuning of the characteristics of the bumps and valleys, the accuracy increases. 1-D models have been used, within the context of drug delivery, to predict the effect of the morphology of the media on the diffusion coefficients. Michaels,28,29 for example, used a two-domain, 1-D model to predict the effect of the reduction of area available for the transport of drugs by using species conservation principles and steady-state conditions. Trinh et al.30 used a similar approach to investigating the effect of the modified morphology of the gel on the effective mobility for several situations with and without hindered transport. The results led to analytical expressions qualitatively similar to those of Zimm27 but with the addition of the effect of hindrance on the coefficients. Other contributions also31-33 made use of 1-D models to predict either the mobility ratio or the diffusion ratio. In addition, Boyack and Giddings32 used similar types of approaches for their analysis of porous membranes but introduced a concept borrowed from the theory of catalysis, the tortuosity factor in addition to the constriction concept. In fact, they express the reduced effective mobility as the ratio between the constriction and the tortuosity factors. However, caution must be exercised on the general validity of these results because most of them assumed that both diffusion and convection were important, as opposed to electromigration. This situation is most likely to be valid for low or moderate field values. For regimes of high values of the electrical field, transport of the polyelectrolyte would be dominated by the electromigration effects. In 1991, Zimm34 used a “tube”-based model with two interconnected domains, i.e., a sequence of large pores or lakes connected by narrow straits. The idea is rooted in the “sliplink” concept introduced by Doi and Edwards in 1986.35 The model helped to understand a number of important aspects of the physics of transport of a flexible polyelectrolyte (DNA) in gel electrophoresis. A very useful analysis of the basic aspects of the model and their physical implications as well as a discussion of other similar models can be found in work by Viovy15 (in particular, see section VII.B). 2.3. Scaling Arguments. A very useful description of the motion of the polyelectrolyte inside gels can be obtained by scaling the dynamics of the molecule with respect to the scales related to the direction of motion under an applied electrical field. Slater (one of the pioneers of this concept) explained simply36 that if one assumes that the polyelectrolyte molecule covers several pores of the gel, then the gel fibers will restrict the lateral motion of the molecule. For this case, “the short length scale dynamics of the chain inside the tunnel” is unimportant to control the net motion of the molecule in the direction of the field. This net motion has been referred to as “reptation”. In order to have a historical perspective of the development of the model and implications as well as its relationship to other significant contributions, the reader is referred to a very didactic introduction by Slater.36 This author made the connection to two other very useful overviews of efforts on the subject.17,19 An in-depth analysis of the calculation


and an estimation of the scaled quantities used in the approach can be found in work by Viovy15 (in particular, see section V). [There are other scaling efforts for the transport of particles in separation media based on continuum mechanics. The reader is referred to Slater et al. for a review.37 2.4. Direct Computational Simulation Approaches. We echo here a suggestion made by Viovy15 to group other contributions that help to understand the motion of different polyelectrolyte molecules inside gels (see section VII.A). Researchers have used several different approaches to capture the dynamics of the polyelectrolyte molecule in a variety of domains, including Monte Carlo (MC), Brownian dynamics (BD), Langevin dynamics (LD), molecular dynamics (MD), and Lattice Boltzmann (LB), among others. A very up-to-date review of the methods and results may be found in work by Slater et al.37 and earlier contributions (until 1990) may be found in work by Cantor and Lim.38 We include some of these early contributions in Table 3, and some other relevant contributions are given in Thormann et al.39 2.5. Directions in Nanocomposite Electrophoresis Model Development. The conceptual problem of understanding the properties and transport in nanocomposite hydrogels includes three different but complementary modifications to the previously described models: (a) a description of the distribution of nanoparticles within the gels; (b) a description of the motion of the buffer solution that may include an electroosmotic effect for nanoparticles with surface charges; (c) a description of the motion of the polyelectrolyte molecule inside the new gel material for a given application. Note that the latter description might include analyte motion when the nanochannels are narrow enough to develop overlap of the electrical double layers. Regarding the description of the nanoparticle distribution, Hill40 has presented a related analysis for charged colloidal particles embedded in an uncharged hydrogel, where the colloidal particle can move. This effort coupled the electrokinetic-based displacement of the colloidal particle with the elastic deformation of the equations for the gel. The gel media are treated as elastic, incompressible Brinkman media. Hill’s effort is motivated by the experimental work reported by Matos et al.41 that has shown that the addition of these particles enhances the electroosmotic flow inside the gel. Some of the conclusions reported by Hill include the effects of the nanoparticle surface charge, nanoparticle size, Young’s modulus of the gel skeleton, and ionic strength of the buffer solution upon displacement of the nanoparticle. In neither case was the motion of a biomolecular analyte described, only the phases of the gel matrix, buffer, and nanoinclusion. • The key obstacle for future research efforts here remains the practical aspects of characterizing nanoparticle dispersion on a variety of scales and adding the movement of the analyte. Quantitative descriptions of the interparticle distance will add to its computational relevance, as well as descriptions of how environmental conditions alter the surface charge. The transport of electrolytes, i.e., the buffer solution, has also been discussed by Hill42 using concepts based on continuum mechanics. [Aspects related to the nanoscale transport of DNA have been reviewed by Baldessari and Santiago, and an excellent discussion of fundamental, potential problems and applications may be found in Schoch et al. The reader should be cautious on application of these concepts to nanocomposite gels because the conditions and characteristics of such complex structures may be unique.43,44] He reports that diffusion and electromigration are shown to be independent of convection and, therefore, independent of the Darcy (polymer gel) permeability.

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Moreover, optimal pumping inside the gel is favored by thin membranes with large cross sections, a high inclusion volume fraction, and low electrolyte conductivity. The behavior of the system to an imposed bulk electrolyte concentration gradient was reported by Hill.45 In addition, the electroosmotic pumping velocity from flux enhancements has been determined from a mathematical model developed by Hill.46 After a comparison of the theory and experiments, it was concluded that the macroscale properties are linked to the microstructure, which needs further characterization. • With regard to the motion of the buffer, Hill concluded that future experiments might report the conductivity of hydrogels with and without inclusions to aide in the theoretical predictions and that silica nanoinclusions have been demonstrated to increase the overall free volume of the polymer membranes, and therefore characterization of the free volume state of the nanocomposite would be important.47 The present authors additionally see that a description of the buffer flow regimes in nanochannels would be important in determining the role of electroosmosis in polyelectrolyte transport behavior. Finally, the assessment of how various morphologies can enhance separation may be informed by using idealized models for the pore geometry versus the different types of polyelectrolytes, as indicated in Figure 2. Rigorous analysis of the hydrodynamics in such a pore by the lubrication approximation coupled with an area-averaged approach was used to predict the optimal time of separation in an idealized conical and divergent section by Pascal et al.48 Moreover, Simhadri et al.49 have shown through simulation that the most significant effect of variation of the angle of divergence at the entrance to a pore seems to be located within the range of up to 20°.50 In addition, Thompson et al. have shown intriguing experimental behaviors for protein mobility through nanocomposite gels, and they were able to predict this behavior (partially) by using membranebased models.51 These have been motivated by experimental results that show a clear enhancement of the separation of proteins by the presence of nanoparticles modifying the gel morphology.52,53 • Modeling efforts that describe how proteins move through single nanochannels as a function of environmental conditions such as the isoelectric point could aide in predicting behavior in systems of nanochannels (which the nanocomposite represents) provided some scaling arguments are developed to connect the two areas of research. These results are quite relevant for the separation of biological molecules by electrophoresis, and future efforts are expected to provide significant new opportunities for efficient native-type separations. 3. Morphological Changes in Gels Given the theoretical emphasis on the importance of morphology, a review of experimental developments in nanocomposite morphologies follows for comparison. Here we quickly give some details on the templating approach mentioned previously but focus this review finally on contributions regarding the effects of the addition of nanoparticles on gel morphologies. For the case of templating, early work54-56 showed promising results in enhancing the protein separation by manufacturing a gel templated by biomacromolecules, such as DNA, of selected sizes. The material may be synthesized by adding the DNA molecules and proceeding to polymerization until completion. The DNA molecules are then removed by electrophoresis until practically none of them are left inside the gel matrix. What is

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left behind is presumed to be pores of the exact dimensions of the DNA template. Protein electrophoresis results have shown enhanced separation when using templated gels. An idealized “composite” matrix with two void size distribution populations was hypothesized to explain the possible reason behind this important behavior.30 Other potential templating materials were also reported,56-59 confirming the trends. These results offer a “proof of concept” for the particle-mediated change of the gel morphology as a potentially unique factor for controlling and, more importantly, tailoring electrophoresis separations in gels. However, the templating advances described may suffer during scale-up because of difficulties in handling. For the case of the addition of nanoparticles, the following sections review advances in hydrogel-nanoparticle systems that have demonstrated a potential in biotechnological applications. These are outlined by the type of nanoinclusion, as well as the chemical composition of the polymer. Table 2 summarizes these different types of nanocomposite gels and presents selected applications. 3.1. Poly(acrylamide) (PAM)/Clay Nanocomposite Hydrogels. For PAM-based nanocomposites, the structure of the polymer backbone is vinylic in nature, with amide groups branching outward. Poly(N-isopropylacrylamide) (PNIPAM) differs in that the hydrophilic amide branches are now terminated with hydrophobic isopropyl groups, and PNIPAM exhibits a reversible phase transition in water at a very easily accessed lower critical solution temperature (LCST) of about 33 °C.60 Phase transitions for such hydrogels can be responsive to various external stimuli including temperature, ionic strength, pH, etc.61 Sershen et al. have even developed a PAM/gold (Au) nanoparticle nanocomposite, which forms a microfluidic “valve”, and the transition is activated by either green light or IR radiation.62 Additional properties (beyond pore morphology) for PAM/clay nanocomposites have been reviewed recently by Haraguchi.63 PAM-based nanocomposites formed with organoclay nanoparticles have attracted researchers to understand the nature of the organoclay as a cross-linker in such systems as well as the clay’s influence on thermoresponsivity, swelling, and elasticity. Synthesis of the nanocomposite gels initially requires a stable uniform dispersion of exfoliated clay platelets in a solution that contains monomers and other reagents. Free-radical polymerization can then be initiated on the clay disks, thus forming a polymer brush. The brush polymers grow longer, interconnecting multiple clay particles, and finally form a cross-linked polymer network.64-66 Success with this protocol has been reported using a synthetic hectorite, Laponite XLG. A comparison of a Laponite cross-linked PNIPAM nanocomposite (NC) gel with the organically cross-linked (OR) gel has been reported by Haraguchi and co-workers.67,68 The NC gels had a uniform network structure and maintained their transparency even when the clay content was increased. Contrary to this, the OR gel transparency decreased markedly when the organic cross-linker/NIPA monomer ratio exceeded 5 mol %. Network inhomogeneities were observed to be the cause of the OR gel’s poor transparency. For NC gels, the deswelling kinetics gradually decreased as the clay content was increased, a result opposite of that for OR gels.69 The OR gels demonstrated a high absolute cross-link density compared to the NC gels as well as a broader distribution of chain lengths; hence, OR gels have a population of short inter-cross-link distances. OR gels were easily fractured. On the other hand, NC gels having a homogeneous distribution of cross-linker avoid localization of stress under deformation and show greater strengths and resistance to fracture.

The structure and dynamics of the PNIPAM/Laponite XLG nanocomposite gels were investigated by Shibayama et al. using small-angle neutron scattering, small-angle X-ray scattering, and dynamic light scattering.70 They observed that the chain dynamics for the composite hydrogels were similar to the chain dynamics of chemically cross-linked PNIPAM gels. However, in terms of the structure, the PNIPAM chains were found to be anchored to the clay platelets. Nie et al. has reported on the formation of PNIPAM/Laponite RDS hydrogels. Laponite RDS is different from Laponite XLG in that the edges of the clay platelets are modified to achieve a negative surface charge. PNIPAM/Laponite RDS nanocomposites have been studied by means of mechanical, swelling ratio, and static light scattering measurements.71 These authors observed two correlation lengths irrespective of the preparation conditions, one around 200-250 nm and another at several tens of nanometers. They concluded that the clay particles act as cross-links with an average functionality of around 50 and that the large-scale structure was formed as a result of the kinetically controlled rearrangement of the clay particles during formation of the gels. The network structure, spatial inhomogeneity, and chain dynamics have been investigated by dynamic light scattering.61 Thermal fluctuations increased strongly with an increase in the Laponite RDS content and decreased with an increase in the monomer concentration. They concluded that the residual mobility of the clay particles was suppressed by formation of the network and that the chain dynamics was independent of the cross-linker concentration. PAM/Laponite RDS nanocomposite gels were synthesized by Okay and Oppermann using free-radical polymerization of acrylamide in an aqueous clay dispersion with and without the presence of chemical cross-linker, and the resulting gels have been investigated by rheometry.72 At clay concentrations of 5% or above, the nanocomposite hydrogels with or without a chemical cross-linker were much more viscous than the conventional hydrogel, which indicated that the rubber elasticity of hydrogels was mainly due to clay. Further, the loss factor, tan δ, showed a markedly stronger dissipation mechanism for clay-based composites, an observation in accord with the rather large correlation lengths found by Nie et al.71 Further dynamic light scattering showed that clay acts as both a cross-linker and an ionic component during gel formation. PNIPAM/montmorillonite (MMT) organoclay nanocomposites with an enhanced rate of water release and greater swelling ratios have been synthesized by manipulating the interfacial chemistry between the clay particles and hydrogel as reported by Liang et al.73 PNIPAM/MMT aerogel nanocomposites were formed by Bandi et al. in the presence of different cross-linking agents by in situ free-radical polymerization of the NIPAM monomer in the preformed clay aerogel monolith. The advantage of using a clay aerogel as the inorganic filler is that no exfoliation step is necessary. The resulting nanocomposite maintained its structural integrity and retained its LCST.64 The cross-linking agents used included ethylene glycol dimethacrylate, epoxidized soybean oil acrylate, and (methacryloxypropyl)polysilsesquioxane. Churochkina et al. reported nanocomposite behavior when neutral, slightly charged cationic and anionic PAM hydrogel copolymers were formed with Na-MMT.74 The copolymers included an anionic monomer (2-acrylamido-2-methyl-1-propanesulfonic acid) or a cationic monomer (diallyldimethylammonium chloride). The resulting nanocomposites all had high water absorption characteristics attributed to domains of clay particles, which retain water. The mechanical properties were


also improved. The anionic PAM copolymer gel elastic modulus increased, accompanied by maintenance of its degree of swelling, an interesting balance of properties. 3.2. Poly(vinyl alcohol) (PVA)/Clay Nanocomposite Hydrogels. While PVA is not generally used for protein separations, other biomedical applications have been reviewed by Peppas et al.60 Liu and Hoffman showed that hydrogel composites can be formed from PVA amphiphilic polymers combined with synthetic saponite clay.75 The binding interactions of PVA/saponite were studied by adding cationic and anionic surfactants to the clay. Small amounts of cationic surfactant (tetradecyltrimethylammonium bromide) bound to the surface of the clay made the clay hydrophobic, and the polymer was thought to now bind better to the clay surface in this complex four-component system. However, as the cationic surfactant concentration was increased further, the surfactants bound to PVA make it more hydrophilic, resulting in less polymer binding to the clay and a breakdown of the cross-link network. At an even higher cationic concentration, flocculation of the clay began to occur. Anionic surfactant sodium dodecyl sulfate adsorbed on PVA and rendered it hydrophilic, thereby breaking up polymer-clay interactions and breaking down the network. 3.3. PAM/Silica Nanoparticle Nanocomposite Hydrogels. Matos et al.41 used spherical silica inclusions (7-15 nm) to dope a PAM gel. The resulting nanocomposite gel enhances the mass transfer of solutes by generating electroosmotic flows within the gel, which improves the performance of biosensors. Because electrophoresis requires a charge on the solute, it cannot be used to drive neutral molecules. The method developed by Matos et al. allows for the mass transfer of neutral solutes through the hydrogel by generating electroosmotic flows in the vicinity of the charged silica inclusions. 3.4. PAM/Carbon Nanotube (CNT) Nanocomposite Hydrogels. Huang et al.76 synthesized a novel nanocomposite PAM gel containing functionalized CNTs. CNT’s were changed from hydrophobic to hydrophilic by grinding in the presence of Triton X. These nanocomposite gels have been shown to improve the separation of apolipoprotein A-I and complement C3 proteins because of interaction of CNT-triton conjugate with the proteins, which was termed a “matrix assist”. 3.5. PAM/PNIPAM Microparticle Composite Hydrogels. Temperature-sensitive PNIPAM microgels were incorporated into a nonresponsive PAM hydrogel matrix at two different temperatures to address whether or not the temperaturedependent swelling behavior of the microparticles is retained when constrained in the composite.77 The size of the microparticles was obtained by small-angle neutron scanning measurements. The resulting hydrogel had thermosensitive optical properties, and swelling/shrinking of the microgel particles was not hindered by the macroscopic hydrogel networks surrounding them, irrespective of different cross-linking densities. Confocal fluorescence microscopy analysis verified that the gel matrix does not significantly influence the temperature sensitivity of the embedded beads. Zhang and Chu have reported microparticles consisting of a PNIPAM/poly(ethylene glycol) diacrylate microgel added to a solution of NIPAM monomer and cross-linker to form a microgel-impregnated PNIPAM hydrogel.78 The pore size of the resulting composite hydrogel was reduced as the concentration of the microgel was increased, as characterized by scanning electron microscopy. Cavities of 30-40 µm were the spaces occupied by the microparticles and were sporadically distributed in the hydrogel matrix network. The composite had excellent

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compressive strength and also retained its LCST behavior, as determined by differential scanning calorimetry. Because of the presence of microgel particles, the composite showed faster response rates and higher equilibrium swelling ratios, depending on the microparticle inclusion concentration. 3.6. Polymer/Metal Nanoparticle Nanocomposite Hydrogels. Composite materials consisting of nanoparticulate Au, silver (Ag), and iron (Fe) in a polymer hydrogel matrix have potential applications in photonics, catalysis, electronics, optics, and biomedicine79 because of novel optical, magnetic, and electronic behaviors. However, here we focus on unique morphologies relevant to bioseparations/sieving. A novel technique to synthesizing a PNIPAM/Au nanoparticle composite has been described by Wang et al.80 Instead of physically combining the nanoparticles prior to gelation, these authors reduced the Au in situ after gelation. The LCST for this copolymerized nanocomposite moved from the control value of 32 °C to a composite value of 45 °C. Swelling ratios improved from values of about 1 to greater than 3.5. Osmotic swelling was attributed to a dependence on the presence of the immobilized charge in the network, which was increased in this case by the large surface area of the attached nanoparticulate Au. Another approach to preparing a PNIPAM/Au nanocomposite was to functionalize the surface of Au nanoparticles with an allylmercaptan, thereby exposing vinyl groups to react with the NIPA monomer81 during polymerization. The swelling degree decreased for the composite because the nanoparticulate Au participated as a cross-linker, similar to the MMT clays discussed earlier. The electrical conductivity of the deswollen hydrogel increased by 2 orders of magnitude. Pardo-Yissar et al. have described a novel “breathing” mechanism for the formation of Au nanoparticles embedded in an electropolymerized PAM hydrogel.82 While no unique morphologies/pore structures were necessarily described here, certainly this technique holds promise for producing gradients of nanoparticles in hydrogels. In this case, the swelling/ deswelling cycle, or the “breathing”, is accomplished by alternation between a solvent/antisolvent system of water and acetone. The final composite with Au nanoparticles embedded can be switched between electrically communicating/noncommunicating states through deswelling/swelling. Kazimierska and Ciszkowska produced Au-nanoparticleembedded PNIPAM hydrogels, with Au ranging from 2.7 to 18 nm in diameter. They found that diffusion coefficients for 1,1′-ferrocenedimethanol (Fc(MeOH)2) in gels were lower than those in aqueous solutions but were not affected by the presence of the Au nanoparticles. Also, the matrix transition temperature was unaffected by the Au nanoparticles.83 Mohan et al. have reported a facile synthesis of poly(NIPAMco-sodium acrylate) with in situ reduction of Ag nanoparticles.84 The resulting Ag nanoparticles (3 nm) were highly stabilized by the sodium acrylate units in the hydrogel networks, with some analogy to the Au nanocomposites previously discussed by Wang et al.80 These authors report very tight control of the nanoparticle size by changing the cross-linker content, with smaller nanoparticles being formed at higher cross-linker densities. 3.7. Polymer/Magnetic Nanoparticle Nanocomposite Hydrogels. Schmidt has published a recent review of Fe-containing thermoresponsive hydrogels, in which materials of interest to bioseparations are most often based on ferrous sulfate (FeSO4) and PNIPAM.85 Temperature-sensitive properties of these particles have been used to reversibly disperse the core-shell

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particles and to reversibly adsorb or desorb proteins because of hydrophobic/hydrophilic switching of PNIPAM above and below the LCST. Thus, the possibility that certain core-shell nanoparticles introduced into a hydrogel matrix may affect both the morphology and affinity in some coupled manner is an important consideration,76,86 just as Matos et al. have described coupled effects on both the morphology and electroosmotic behavior.41,46 Schmidt also reviewed how the localized gel surrounding a nanoparticle can be heated using an external magnet and how these thermal effects in the presence of a thermosensitive polymer produce morphological changes.85 Lao and Ramanujan have additionally given some useful details of the heating characteristics of ferrogels formed from PAM.87 Lastly Schmidt mentions several applications in which core-shell thermosensitive particles can aid separations for proteins and cells; the thermosensitive nature of the materials provides for reversible adsorption/desorption of the analyte, and the magnetic nature provides for separation.85 As a side note to Fe-based nanoparticles in hydrogels, the ubiquitous core-shell morphology and magnetic heatability of these materials certainly opens up some unusual possibilities for the tunable morphology of the composite. However, we might also consider the usefulness of the geometric opposite of core-shell morphology (with a thermoresponsive shell), this being referred to in the literature often as a “gate” (with a thermoresponsive core). Yameen et al. have described one example of a “gate” morphology, in which a polyimide membrane matrix is resistive to analyte flow, and the analytes move instead through etched pores coated with thermosensitive PNIPAM brush gates.88 We suggest that one could imagine localized heating using Fe nanoparticles, which would activate the gate. 4. Balance of Properties for Nanocomposite Hydrogels Nanocomposite hydrogels are used in a large number of applications where properties beyond separation are critical. For example, in some medical diagnostics, the gel should be clear because detection protocol often requires clarity. Further, the gel must be mechanically tough because many staining techniques depend on lifting of the gel. Therefore, the following section reviews some examples where synthetic hydrogelnanoparticle systems exhibit dramatic mechanical property enhancements, making them excellent candidates to replace the current gels largely used in today’s protocols. Note that improvements in elongation are our focus because this property can be related to mechanical toughness. 4.1. PAM/Clay Nanocomposite Hydrogels. For PNIPAM hydrogels modified with a synthetic hectorite (Laponite XLG), Haraguchi et al.63,65,67,69 have reported significantly enhanced strength and fracture energies. Note that the organic cross-linker [e.g., bis(acrylamide)] was not used in the nanocomposite materials. The nanocomposite stress-strain curve showed a ductile yield and stress hardening behavior. Tensile stress at break for a “NC20” composite was almost 1000 kPa, where the corresponding nonmodified gel broke at about 9 kPa. The fracture energies showed a nearly monotonic increase with the Laponite mole percent. Up to 3.3 J of fracture energy was measured for “NC20” versus a 2500 times lower value for the neat hydrogel. The compressive properties were also reported by Haraguchi and Song.89 Some composites fractured beyond 80% strain. It was found that, in terms of the trade-off between the compressive strength and modulus, gels cross-linked by both nanoparticles and bis(acrylamide) showed enhanced compressive properties. Some evidence was noted of bis(acrylamide) dis-

tributing preferentially in the immediate vicinity of the clay platelets. The transparency for the NC3 and NC4 gels was very low above the LCST of PNIPAM, just as for the neat material. For NC7 and higher loadings, the transparency difference above and below the LCST was not very marked. They concluded that thermal molecular motion was restricted at the higher loadings, particularly for NC15 or greater, and consequently the thermosensitivity of these hydrogels was lost at those upper loadings. However, if thermosensitivity was not required, the higher loadings offered marked transparency even at high temperatures. PNIPAM hydrogels cross-linked with both bis(acrylamide) and MMT organoclay (Cloisite 30B) were investigated by Lee and Fu.90 The shear modulus increased from 6.5 MPa to a modest 11.4 MPa with 15% organoclay. Ma et al.91 have reported on an extension of the Haraguchi system, in which sodium carboxymethylcellulose is incorporated along with PNIPAM/Laponite XLG in a semi-IPN, and obtained elongations as high as 1600%. The IPN gels swelled faster than PNIPAM/clay at pH 7.4 and slower at pH 1.2. Zhu et al. have reported the mechanical properties for nanocomposites made with both PNIPAM and PAM combined with Laponite XLS.66 These authors report that the XLS clay offers some preparation advantages over Laponite XLG including ease of mixing. The PAM/Laponite XLS hydrogels achieved 325 kPa of stress at break with 10% (w/w) clay and 20% (w/ w) polymer. The elongation for this material was 1672%, a remarkable increase. In addition, the elastic recovery was 96% and the hysteresis ratio was 20%. Zhang et al.92 found that the mechanical properties of PAM/Laponite XLS gels increased when it was heat treated at 40 °C for 5-20 days. They postulated that some of the functional groups in the polymer were interacting with the clay surface and that this interaction decreased with heat treatment. Swelling ratios were seen to increase remarkably, consistent with their postulation. The tensile elongation was seen to increase from 1814 to 2362% and the strength at break from 120 to 219 kPa. Haraguchi et al.93 have examined such properties for a structural analogue of PAM, poly(N,N-dimethylacrylamide) (PDMAM), with Laponite XLG. The DMAM monomer is soluble at all proportions in water, and the polymer does not exhibit LCST behavior in water. Again, large elongations, up to 1500%, were noted. 4.2. PAM/CNT Nanocomposite Hydrogels. The mechanical properties of single-walled CNT (SWNT) hydrogel composites were qualitatively discussed by Makamba et al.94 in a microfluidic application that involved protein separations. The SWNTs were functionalized with a workup that eventually yielded a SWNT/poly(ethylene glycol) acrylate material. The acrylamidebased hydrogel composite was photopolymerized in a microfluidic channel. They reported that the nanocomposite was robust enough to perform electrophoresis for 5 days, whereas the control was swept out of the channel on the first water flush. 4.3. Polymer/Metal Nanoparticle Nanocomposite Hydrogels. A recent example of a very complex morphology involving ferrogels is described by Ma and Zhang,95 who give some additional details concerning the dynamic complex modulus and gelation kinetics. They varied the composition of the ferrogels, with the constituents being a poly(ethylene glycol)/poly(caprolactone) block copolymer, a cyclic oligosaccharide, and iron oxide nanoparticles. 4.4. Directions in Nanocomposite Hydrogel Material Development for Electrophoresis. A multiplicity of new materials in nanocomposite hydrogels has been reviewed, and while applications are ubiquitous, few are specifically designed


for electrophoretic applications. In particular, we have seen that hydrogels with silica inclusions (which have a surface charge) can potentially take advantage of electroosmosis, with applications in mixing or even facilitated transport of neutral analytes. Other novel separations envisioned include smart or tunable nanoparticle-analyte effects, such as externally activated “gates”. The formation of physically cross-linked gels in place of covalent cross-links might lead to postprocessing of the gel toward the facile collection of native biomolecular samples. Certainly these physically cross-linked gels exhibited dramatic improvements in elongation, related to their ease of handling. As a final note, one difficulty often reported by medical diagnosticians is that hydrogels might have a short shelf life or a heterogeneous cross-link density or are difficult to reproduce. Certainly advances in nanocomposite gel development hold the potential to answer these problems. 5. Concluding Remarks Nanoinclusions have been shown to play an important role in determining the hydrogel morphology, as demonstrated by a survey of nanocomposite hydrogels keyed to both the nanoparticle and matrix. Further, it is clear from the review of polyelectrolyte transport modeling for hydrogels that the presence of inclusions will affect both the analyte mobility and diffusion in a complex manner. The role of the nanoparticle in the composite gel also affords improved handling characteristics, which may be the key property determining early adoption. Thus, interdisciplinary efforts in both modeling and experimental polyelectrolyte transport studies can yield novel efficient separation avenues from this new class of materials. Acknowledgment We are grateful for numerous discussions with doctoral students Jennifer A. Pascal and Jeffrey Thompson from our research group and to Dr. Robert Sanders (Director of Research at TTU) for his input on the clinical diagnostic aspects. P.E.A. is indebted to the stimulating environment of the American Electrophoresis Society and, in particular, to Dr. Nancy Stellwagen, Dr. David Garfin, Dr. Nancy Kendrick, Dr. Victor Ugaz, Dr. Neal Ivory, and Dr. Sharon Sauer. Conversations with Dr. Gary Slater and members of his group have always been motivating. Financial support from the Center for Manufacturing Research at TTU, the Diversity Fellowship Program sponsored by the Office of Research at TTU, and the Universidad Catolica del Norte, Antofagasta, Chile, is greatly appreciated. List of Abbreviations Used AM ) acrylamide Au ) gold CNT ) carbon nanotube LCST ) lower critical solution temperature MMT ) montmorillonite NC ) nanocomposite OR ) organically cross-linked PAM ) poly(acrylamide) PDMAM ) poly(N,N-dimethylacrylamide) PEG-DA ) poly(ethylene glycol) diacrylate PNIPAM ) poly(N-isopropylacrylamide) PVA ) poly(vinyl alcohol) SA ) sodium acrylate

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ReceiVed for reView February 18, 2010 ReVised manuscript receiVed July 20, 2010 Accepted July 26, 2010 IE1003762

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