Biocatalytic Self-Assembly on Magnetic Nanoparticles.

Combining (bio)catalysis and molecular self-assembly provides an effective approach for the production and processing of self-assembled materials by exploiting catalysis to direct the assembly kinetics and hence controlling the formation of ordered nanostructures. Applications of (bio)catalytic self-assembly in biologically interfacing systems and in nanofabrication have recently been reported. Inspired by self-assembly in biological cells, efforts to confine catalysts on flat or patterned surfaces to exert spatial control over molecular gelator generation and nanostructure self-assembly have also emerged. Building on our previous work in the area, we demonstrate in this report the use of enzymes immobilized onto magnetic nanoparticles (NPs) to spatially localize the initiation of peptide self-assembly into nanofibers around NPs. The concept is generalized for both an equilibrium biocatalytic system that forms stable hydrogels and a nonequilibrium system that normally has a preset lifetime. Characterization of the hydrogels shows that self-assembly occurs at the site of enzyme immobilization on the NPs to give rise to gels with a "hub-and-spoke" morphology, where the nanofibers are linked through the enzyme-NP conjugates. This NP-controlled arrangement of self-assembled nanofibers enables both remarkable enhancements in the shear strength of hydrogel systems and a dramatic extension of the hydrogel stability in the nonequilibrium system. We are also able to show that the use of magnetic NPs enables the external control of both the formation of the hydrogel and its overall structure by application of an external magnetic field. We anticipate that the enhanced properties and stimuli-responsiveness of our NP-enzyme system will have applications ranging from nanomaterial fabrication to biomaterials and biosensing.

New antifouling platform characterized by single-molecule imaging.

Antifouling surfaces have been widely studied for their importance in medical devices and industry. Antifouling surfaces mostly achieved by methoxy-poly(ethylene glycol) (mPEG) have shown biomolecular adsorption less than 1 ng/cm(2) which was measured by surface analytical tools such as surface plasmon resonance (SPR) spectroscopy, quartz crystal microbalance (QCM), or optical waveguide lightmode (OWL) spectroscopy. Herein, we utilize a single-molecule imaging technique (i.e., an ultimate resolution) to study antifouling properties of functionalized surfaces. We found that about 600 immunoglobulin G (IgG) molecules are adsorbed. This result corresponds to ∼5 pg/cm(2) adsorption, which is far below amount for the detection limit of the conventional tools. Furthermore, we developed a new antifouling platform that exhibits improved antifouling performance that shows only 78 IgG molecules adsorbed (∼0.5 pg/cm(2)). The antifouling platform consists of forming 1 nm TiO2 thin layer, on which peptidomimetic antifouling polymer (PMAP) is robustly anchored. The unprecedented antifouling performance can potentially revolutionize a variety of research fields such as single-molecule imaging, medical devices, biosensors, and others.

Macromolecular shape and interactions in layer-by-layer assemblies within cylindrical nanopores.

Layer-by-layer (LbL) deposition of polyelectrolytes and proteins within the cylindrical nanopores of anodic aluminum oxide (AAO) membranes was studied by optical waveguide spectroscopy (OWS). AAO has aligned cylindrical, nonintersecting pores with a defined pore diameter d(0) and functions as a planar optical waveguide so as to monitor, in situ, the LbL process by OWS. The LbL deposition of globular proteins, i.e., avidin and biotinylated bovine serum albumin was compared with that of linear polyelectrolytes (linear-PEs), both species being of similar molecular weight. LbL deposition within the cylindrical AAO geometry for different pore diameters (d(0) = 25-80 nm) for the various macromolecular species, showed that the multilayer film growth was inhibited at different maximum numbers of LbL steps (n(max)). The value of n(max) was greatest for linear-PEs, while proteins had a lower value. The cylindrical pore geometry imposes a physical limit to LbL growth such that n(max) is strongly dependent on the overall internal structure of the LbL film. For all macromolecular species, deposition was inhibited in native AAO, having pores of d(0) = 25-30 nm. Both, OWS and scanning electron microscopy showed that LbL growth in larger AAO pores (d(0) > 25-30 nm) became inhibited when approaching a pore diameter of d(eff,n_max) = 25-35 nm, a similar size to that of native AAO pores, with d(0) = 25-30 nm. For a reasonable estimation of d(eff,n_max), the actual volume occupied by a macromolecular assembly must be taken into consideration. The results clearly show that electrostatic LbL allowed for compact macromolecular layers, whereas proteins formed loosely packed multilayers.

Mussel-inspired silver-releasing antibacterial hydrogels.

A silver-releasing antibacterial hydrogel was developed that simultaneously allowed for silver nanoparticle formation and gel curing. Water-soluble polyethylene glycol (PEG) polymers were synthesized that contain reactive catechol moieties, inspired by mussel adhesive proteins, where the catechol containing amino acid 3,4-dihydroxyphenylalanine (DOPA) plays an important role in the ability of the mussel to adhere to almost any surface in an aqueous environment. We utilized silver nitrate to oxidize polymer catechols, leading to covalent cross-linking and hydrogel formation with simultaneous reduction of Ag(I). Silver release was sustained for periods of at least two weeks in PBS solution. Hydrogels were found to inhibit bacterial growth, consistent with the well-known antibacterial properties of silver, while not significantly affecting mammalian cell viability. In addition, thin hydrogel films were found to resist bacterial and mammalian cell attachment, consistent with the antifouling properties of PEG. We believe these materials have a strong potential for antibacterial biomaterial coatings and tissue adhesives, due to the material-independent adhesive properties of catechols.

An experimental-theoretical analysis of protein adsorption on peptidomimetic polymer brushes.

Surface-grafted water-soluble polymer brushes are being intensely investigated for preventing protein adsorption to improve biomedical device function, prevent marine fouling, and enable applications in biosensing and tissue engineering. In this contribution, we present an experimental-theoretical analysis of a peptidomimetic polymer brush system with regard to the critical brush density required for preventing protein adsorption at varying chain lengths. A mussel adhesive-inspired DOPA-Lys (DOPA = 3,4-dihydroxy-phenylalanine; Lys = lysine) pentapeptide surface grafting motif enabled aqueous deposition of our peptidomimetic polypeptoid brushes over a wide range of chain densities. Critical densities of 0.88 nm(-2) for a relatively short polypeptoid 10-mer to 0.42 nm(-2) for a 50-mer were identified from measurements of protein adsorption. The experiments were also compared with the protein adsorption isotherms predicted by a molecular theory. Excellent agreements in terms of both the polymer brush structure and the critical chain density were obtained. Furthermore, atomic force microscopy (AFM) imaging is shown to be useful in verifying the critical brush density for preventing protein adsorption. The present coanalysis of experimental and theoretical results demonstrates the significance of characterizing the critical brush density in evaluating the performance of an antifouling polymer brush system. The high fidelity of the agreement between the experiments and molecular theory also indicate that the theoretical approach presented can aid in the practical design of antifouling polymer brush systems.

Layer-by-Layer Assemblies in Nanoporous Templates: Nano-Organized Design and Applications of Soft Nanotechnology.

The synergistic combination of layer-by-layer (LbL) assembly and nanoporous membrane templating has greatly facilitated the creation of complex and functional nanotubular structures. The approach takes advantage of both the new properties conferred by assembling diverse LbL building blocks and the tight dimensional control offered by nanotemplating to enable new functionalities that arise from the highly anisotropic "one-dimensional" LbL-nanotube format. In this review, we aim to convey the key developments and provide a current snap-shot of such templated LbL nanoarchitectures. We survey recent developments that have enabled the assembly of polymers, biomolecules and inorganic nanoparticles "à la carte", via electrostatic, covalent and specific (bio)recognition interactions. We also discuss the emerging mechanistic understanding of the LbL assembly process within the nanopore environment. Finally, we present a diverse range of LbL nanotube "devices" to illustrate the versatility of the nanotemplated LbL toolbox for generating functional soft nanotechnology.

Mounted nanoporous anodic alumina thin films as planar optical waveguides.

Solid-supported thin films of self-organized nanoporous anodic aluminum oxide (AAO) have been widely employed for the template preparation of nanostructured functional materials. Recently, the use of nanoporous AAO thin films in optical waveguide spectroscopy (OWS) has been explored for high sensitivity, in situ monitoring of processes occurring within these nanoporous templates. In this contribution, we demonstrate a strategy for mounting bulk anodized AAO thin films on heterogeneous solid-supports suitable for waveguide sensing experiments. Unlike conventional preparations of AAO thin films by anodization of vacuum- or electrochemically deposited Al thin films, the full range of techniques available to anodize bulk Al may potentially be applied with the present method. Moreover, we show that AAO thin films mounted on glass substrates can have superior waveguide coupling properties compared to conventionally prepared samples. The nanostructure of the AAO can be well characterized by an EMT-OWS analysis, demonstrated by comparing scanning electron microscopy images of the AAO and the pore dimensions calculated from an effective medium theory (EMT) analysis of the film refractive index measured by OWS. Finally, using a curved metallic substrate as an example, we show that our mounting technique can be used as a general strategy to functionalize objects with nanoporous AAO films.

Polyelectrolyte layer-by-layer deposition in cylindrical nanopores.

Layer-by-layer (LbL) deposition of polyelectrolytes within nanopores in terms of the pore size and the ionic strength was experimentally studied. Anodic aluminum oxide (AAO) membranes, which have aligned, cylindrical, nonintersecting pores, were used as a model nanoporous system. Furthermore, the AAO membranes were also employed as planar optical waveguides to enable in situ monitoring of the LbL process within the nanopores by optical waveguide spectroscopy (OWS). Structurally well-defined N,N-disubstituted hydrazine phosphorus-containing dendrimers of the fourth generation, with peripherally charged groups and diameters of approximately 7 nm, were used as the model polyelectrolytes. The pore diameter of the AAO was varied between 30-116 nm and the ionic strength was varied over 3 orders of magnitude. The dependence of the deposited layer thickness on ionic strength within the nanopores is found to be significantly stronger than LbL deposition on a planar surface. Furthermore, deposition within the nanopores can become inhibited even if the pore diameter is much larger than the diameter of the G4-polyelectrolyte, or if the screening length is insignificant relative to the dendrimer diameter at high ionic strengths. Our results will aid in the template preparation of polyelectrolyte multilayer nanotubes, and our experimental approach may be useful for investigating theories regarding the partitioning of nano-objects within nanopores where electrostatic interactions are dominant. Furthermore, we show that the enhanced ionic strength dependence of polyelectrolyte transport within the nanopores can be used to selectively deposit a LbL multilayer atop a nanoporous substrate.

The effect of fluid flow on selective protein adsorption on polystyrene-block-poly(methyl methacrylate) copolymers.

The effect of fluid flow on protein patterning and the stability of the adsorbed protein array templated by polystyrene-block-poly(methyl methacrylate) (PS-b-PMMA) has been investigated. Protein nanoarrays can be formed on a PS-b-PMMA copolymer surface by physisorption and rinsing with an open flow. The protein arrays inherit the original hexagonal pattern generated by the phase separation of PS-b-PMMA with the proteins only residing on the PS domains. Subsequent analysis of the protein array stability under confined flow field reveals that the array integrity is strongly dependent on the flow velocity and the duration time. The interplay between the intermolecular forces and the hydrodynamic shear force has been discussed in detail. A simplified model has been proposed to explain the site-selective adsorption and protein migration under the shear of fluid flow. This study provides valuable information on the formation mechanism and stability of physisorbed protein nanopatterns under conditions concerning biosensing applications. It also represents an explorative study of protein adsorption under hydrodynamic flow conditions, which may assist in better designs of fluidic devices relevant to protein studies.

Modulation of protein-surface interactions on nanopatterned polymer films.

The introduction of nanoscale features brings with it a high density of surface interface boundaries and effectively introduces an additional boundary material that exhibits properties different from the surrounding surfaces. We systematically varied the feature size of self-assembled polystyrene-block-poly(methyl methacrylate) copolymer nanopatterns from 13 to 200 nm and demonstrated that the basic property of protein adsorption on a nanopatterned surface can be modulated by the length density of surface interfaces present. Protein adsorption on the nanopatterns could be described by a modified adsorption affinity along the surface interface with an effective width on the length-scale of individual proteins. Due to the intrinsic high density of surface interfaces in many polymeric thin film nanopatterns and structures, the interaction of proteins with such interfaces may be of particular relevance to cell-surface studies and to biomaterial and biosensor applications involving nanoscale features.

In situ characterization of N-carboxy anhydride polymerization in nanoporous anodic alumina.

Poly(gamma-benzyl-L-glutamate) (PBLG) has been a popular model polypeptide for a range of physicochemical studies, and its modifiable ester side chains make it an attractive platform for various potential applications. Thin films of Poly(gamma-benzyl-L-glutamate) PBLG were surface grafted within nanoporous anodic alumina (AAO) by surface-initiated polymerization of the N-carboxy anhydride of benzyl-L-glutamate (BLG-NCA). The grafting process was characterized by optical waveguide spectroscopy (OWS), infrared spectroscopy (FT-IR), and scanning electron microscopy (SEM). OWS was able to track the PBLG layer thickness increase in situ, and ex situ FT-IR gave complementary information on the PBLG chain's secondary structure. Transitions in the PBLG growth rate could be correlated with transitions in the polypeptide secondary structure. The emergence of a three-dimensional, anisotropic PBLG morphology within the cylindrical pores of the AAO membrane was also identified as the grafted PBLG average layer thickness increased. Comparison of the PBLG/AAO results with those on a planar silicon dioxide surface indicated that both the conformational transitions and the PBLG nanostructure development could be attributed to the confining geometry within the pores of the nanoporous AAO matrix. The use of a nanoporous AAO matrix, combined with the surface grafting of a thin film of PBLG chains with multiple modifiable side chains, could potentially offer a nanoporous platform with a very high density of functional sites.

Flexible phenylalanine-glycine nucleoporins as entropic barriers to nucleocytoplasmic transport.

Natively unfolded phenylalanine-glycine (FG)-repeat domains are alleged to form the physical constituents of the selective barrier-gate in nuclear pore complexes during nucleocytoplasmic transport. Presently, the biophysical mechanism behind the selective gate remains speculative because of a lack of information regarding the nanomechanical properties of the FG domains. In this work, we have applied the atomic force microscope to measure the mechanical response of individual and clusters of FG molecules. Single-molecule force spectroscopy reveals that FG molecules are unfolded and highly flexible. To provide insight into the selective gating mechanism, an experimental platform has been constructed to study the collective behavior of surface-tethered FG molecules at the nanoscale. Measurements indicate that the collective behavior of such FG molecules gives rise to an exponentially decaying long-range steric repulsive force. This finding indicates that the molecules are thermally mobile in an extended polymer brush-like conformation. This assertion is confirmed by observing that the brush-like conformation undergoes a reversible collapse transition in less polar solvent conditions. These findings reveal how FG-repeat domains may simultaneously function as an entropic barrier and a selective trap in the near-field interaction zone of nuclear pore complexes; i.e., selective gate.