Human Neural Stem Cell Expansion in Natural Polymer Scaffolds Under Chemically Defined Condition.

The maintenance and expansion of human neural stem cells (hNSCs) in 3D tissue scaffolds is a promising strategy in producing cost-effective hNSCs with quality and quantity applicable for clinical applications. A few biopolymers have been extensively used to fabricate 3D scaffolds, including hyaluronic acid, collagen, alginate, and chitosan, due to their bioactive nature and availability. However, these polymers are usually applied in combination with other biomolecules, leading to their responses difficult to ascribe to. Here, scaffolds made of chitosan, alginate, hyaluronic acid, or collagen, are explored for hNSC expansion under xeno-free and chemically defined conditions and compared for hNSC multipotency maintenance. This study shows that the scaffolds made of pure chitosan support the highest adhesion and growth of hNSCs, yielding the most viable cells with NSC marker protein expression. In contrast, the presence of alginate, hyaluronic acid, or collagen induces differentiation toward immature neurons and astrocytes even in the maintenance medium and absence of differentiation factors. The cells in pure chitosan scaffolds preserve the level of transmembrane protein profile similar to that of standard culture. These findings point to the potential of using pure chitosan scaffolds as a base scaffolding material for hNSC expansion in 3D.

Dynamics of Molecular Self-Assembly of Short Peptides at Liquid-Solid Interfaces - Effect of Charged Amino Acid Point Mutations.

Self-organizing solid-binding peptides on atomically flat solid surfaces offer a unique bio/nano hybrid platform, useful for understanding the basic nature of biology/solid coupling and their practical applications. The surface behavior of peptides is determined by their molecular folding, which is influenced by various factors and is challenging to study. Here, the effect of charged amino acids is studied on the self-assembly behavior of a directed evolution selected graphite-binding dodecapeptide on graphite surface. Two mutations, M6 and M8, are designed to introduce negatively and positively charged moieties, respectively, at the anchoring domain of the wild-type (WT) peptide, affecting both binding and assembly. The questions addressed here are whether mutant peptides exhibit molecular crystal formation and demonstrate molecular recognition on the solid surface based on the specific mutations. Frequency-modulated atomic force microscopy is used for observations of the surface processes dynamically in water at molecular resolution over several hours at the ambient. The results indicate that while the mutants display distinct folding and surface behavior, each homogeneously nucleates and forms 2D self-organized patterns, akin to the WT peptide. However, their growth dynamics, domain formation, and crystalline lattice structures differ significantly. The results represent a significant step toward the rational design of bio/solid interfaces, potent facilitators of a variety of future implementations.

Molecular Scale Structure and Kinetics of Layer-by-Layer Peptide Self-Organization at Atomically Flat Solid Surfaces.

Understanding the mechanisms of self-organization of short peptides into two- and three-dimensional architectures are of great interest in the formation of crystalline biomolecular systems and their practical applications. Since the assembly is a dynamic process, the study of structural development is challenging at the submolecular dimensions continuously across an adequate time scale in the natural biological environment, in addition to the complexities stemming from the labile molecular structures of short peptides. Self-organization of solid binding peptides on surfaces offers prospects to overcome these challenges. Here we use a graphite binding dodecapeptide, GrBP5, and record its self-organization process of the first two layers on highly oriented pyrolytic graphite surface in an aqueous solution by using frequency modulation atomic force microscopy in situ. The observations suggest that the first layer forms homogeneously, generating self-organized crystals with a lattice structure in contact with the underlying graphite. The second layer formation is mostly heterogeneous, triggered by the crystalline defects on the first layer, growing row-by-row establishing nominally diverse biomolecular self-organized structures with transient crystalline phases. The assembly is highly dependent on the peptide concentration, with the nucleation being high in high molecular concentrations, e.g., >100 µM, while the domain size is small, with an increase in the growth rate that gradually slows down. Self-assembled peptide crystals are composed of either singlets or doublets establishing P1 and P2 oblique lattices, respectively, each commensurate with the underlying graphite lattice with chiral crystal relations. This work provides insights into the surface behavior of short peptides on solids and offers quantitative guidance toward elucidating molecular mechanisms of self-assembly helping in the scientific understanding and construction of coherent bio/nano hybrid interfaces.

Effect of Degree of Deacetylation of Chitosan/Chitin on Human Neural Stem Cell Culture.

Stem cell therapy and research for neural diseases depends on reliable reproduction of neural stem cells. Chitosan-based materials have been proposed as a substrate for culturing human neural stem cells (hNSCs) in the pursuit of clinically compatible culture conditions that are chemically defined and compliant with good manufacturing practices. The physical and biochemical properties of chitosan and chitin are strongly regulated by the degree of deacetylation (DD). However, the effect of DD on hNSC behavior has not been systematically investigated. In this study, films with DD ranging from 93% to 14% are fabricated with chitosan and chitin. Under xeno-free conditions, hNSCs proliferate preferentially on films with a higher DD, exhibiting adherent morphology and retaining multipotency. Lowering the DD leads to formation of neural stem cell spheroids due to unsteady adhesion. The neural spheroids present NSC multipotency protein expression reduction and cytoplasmic translocation. This study provides an insight into the influence of the DD on hNSCs behavior and may serve as a guideline for hNSC research using chitosan-based biomaterials. It demonstrates the capability of controlling hNSC fate by simply tailoring the DD of chitosan.

Chimeric Peptide-Based Biomolecular Constructs for Versatile Nucleic Acid Biosensing.

Nucleic acid biomarkers hold great potential as key indicators for the diagnosis and monitoring of diseases. Herein we design and implement bifunctional chimeric biomolecules composed of a solid-binding peptide (SBP) domain that specifically adsorbs onto solid sensor surfaces and a peptide nucleic acid (PNA) moiety that facilitates anchoring of antisense oligonucleotide (ASO) probes for the detection of nucleic acid targets. A gold-binding peptide, AuBP1, previously selected by directed evolution to specifically bind to gold, served as the basis for immobilizing nucleic acid probes onto gold substrates. Using surface plasmon resonance (SPR) spectroscopy and quartz crystal microbalance (QCM) analyses, we demonstrate the sequential biomolecular assembly of the heterofunctional solid-binding peptide-antisense oligomer (SBP-ASO) construct onto a sensor surface and the subsequent detection of DNA in an aqueous environment. The effect of steric hindrance on optimal probe assembly is observed, establishing that less packing density results in greater target capture efficacy. In addition, an adsorbed layer of chimeric solid-binding peptide-peptide nucleic acid (SBP-PNA) undergoes viscoelastic changes at the solid-liquid interface upon probe immobilization and DNA target capture, whereby the rigid biofunctional layer becomes more flexible. The dual nature of the chimeric construct is highly amenable to a variety of platforms allowing for both specific recognition and probe immobilization on the sensor surface, while the modular design of the solid-binding peptide-antisense oligonucleotide provides facile functionalization of a wide diversity of solid substrates.

Symbiotic assembly of peptide nano-mosaics at solid interfaces.

The spontaneous co-organization of distinct biomolecules at interfaces enables many of Nature's hierarchical organizations involving both hard and soft materials. Engineering efforts to mimic such hybrid complexes rely on our ability to rationally structure biomolecules at inorganic interfaces. Control over the nanoscale structure of patterned biomolecules remains challenging due to difficulties in controlling the multifarious interactions involved. This work discusses binary peptide assembly as a means to fabricate biomolecular nano-mosaics at graphite surfaces with predictable structures. Distinct peptide-substrate interactions lead to divergent crystallographic growth directions, molecular scale immiscibility, and a symbiotic assembly phenomenon. We present a symbiotic assembly model that accurately predicts the binary assembly structure relying solely on the constituent peptide nucleation kinetics and molar fractions. The ability to tune such biomolecular nano-mosaic structures facilitates the bottom up fabrication of high-density, multifunctional interfaces for nanotechnology.

Bacterial detection using bacteriophages and gold nanorods by following time-dependent changes in Raman spectral signals.

This study attemps to develop bacterial detection strategies using bacteriophages and gold nanorods (GNRs) by Raman spectral analysis. Escherichia coli was selected as the target and its specific phage was used as the bioprobe. Target bacteria and phages were propagated/purified by traditional techniques. GNRs were synthesized by using hexadecyltrimethyl ammonium bromide (CTAB) as stabilizer. A two-step detection strategy was applied: Firstly, the target bacteria were interacted with GNRs in suspensions, and then they were dropped onto silica substrates for detection. It was possible to obtain clear surface-enchanced Raman spectroscopy (SERS) peaks of the target bacteria, even without using phages. In the second step, the phage nanoemulsions were droped onto the bacterial-GNRs complexes on those surfaces and time-dependent changes in the Raman spectra were monitored at different time intervals upto 40 min. These results demonstrated that how one can apply phages with plasmonic nanoparticles for detection of pathogenic bacteria very effectively in a quite simple test.

Effect of Molecular Architecture on Cell Interactions and Stealth Properties of PEG.

PEGylation, covalent attachment of PEG to therapeutic biomolecules, in which suboptimal pharmacokinetic profiles limiting their therapeutic utility are of concern, is a widely applied technology. However, this technology has been challenged by reduced bioactivity of biomolecules upon PEGylation and immunogenicity of PEG triggering immune response and abrogating clinical efficacy, which collectively necessitate development of stealth polymer alternatives. Here we demonstrate that comb-shape poly[oligo(ethylene glycol) methyl ether methacrylate] (POEGMA), a stealth polymer alternative, has a more compact structure than PEG and self-organize into nanoparticles in a molecular weight dependent manner. Most notably, we show that comb-shape POEGMA promotes significantly higher cellular uptake and exhibits less steric hindrance imposed on the conjugated biomolecule than PEG. Collectively, comb-shape POEGMA offers a versatile alternative to PEG for stealth polymer-biomolecule conjugation applications.

Charging of gold/metal oxide/gold nanocapacitors in a scanning electron microscope.

Triangular parallel-plate nanocapacitors were fabricated by a combination of microsphere lithography and physical vapor deposition. The devices were comprised of a 20 nm layer of dielectric material sandwiched between two 20 nm layers of gold. Dielectric materials with a range of relative permittivities were investigated. Charging of the capacitors was probed in a scanning electron microscope (SEM) by monitoring the change in brightness of the images of the devices as a function of time. The time constants, RC, associated with the charging of the capacitors, were extracted from the SEM grayscale data. The resulting average RC values were 248 ± 27 s for SiO2, 70 ± 8 s for Al2O3, 113 ± 80 s for ZnO and 125 ± 13 s for HfO2. These values are consistent with the anticipated RC values based on the resistivities and permittivities of the materials used in the devices and importantly, were measured without the need to attach any wires or leads.

Label-free nanometer-resolution imaging of biological architectures through surface enhanced Raman scattering.

Label free imaging of the chemical environment of biological specimens would readily bridge the supramolecular and the cellular scales, if a chemical fingerprint technique such as Raman scattering can be coupled with super resolution imaging. We demonstrate the possibility of label-free super-resolution Raman imaging, by applying stochastic reconstruction to temporal fluctuations of the surface enhanced Raman scattering (SERS) signal which originate from biomolecular layers on large-area plasmonic surfaces with a high and uniform hot-spot density (>10¹¹/cm², 20 to 35 nm spacing). A resolution of 20 nm is demonstrated in reconstructed images of self-assembled peptide network and fibrilated lamellipodia of cardiomyocytes. Blink rate density is observed to be proportional to the excitation intensity and at high excitation densities (>10 kW/cm²) blinking is accompanied by molecular breakdown. However, at low powers, simultaneous Raman measurements show that SERS can provide sufficient blink rates required for image reconstruction without completely damaging the chemical structure.

Azide photochemistry for facile modification of graphitic surfaces: preparation of DNA-coated carbon nanotubes for biosensing.

A facile, two-step method for chemically attaching single-stranded DNA to graphitic surfaces, represented here by carbon nanotubes, is reported. In the first step, an azide-containing compound, N-5-azido-nitrobenzoyloxy succinimide (ANB-NOS), is used to form photo-adducts on the graphitic surfaces in a solid-state photochemical reaction, resulting in active ester groups being oriented for the subsequent reactions. In the second step, pre-synthesized DNA strands bearing a terminal amine group are coupled in an aqueous solution with the active esters on the photo-adducts. The versatility of the method is demonstrated by attaching pre-synthesized DNA to surfaces of carbon nanotubes in two platforms-as vertically-aligned multi-walled carbon nanotubes on a solid support and as tangled single-walled carbon nanotubes in mats. The reaction products at various stages were characterized by x-ray photoelectron spectroscopy. Two different assays were used to check that the DNA strands attached to the carbon nanotubes were able to bind their partner strands with complementary base sequences. The first assay, using partner DNA strands tethered to gold nanoparticles, enabled the sites of DNA attachment to the carbon nanotubes to be identified in TEM images. The second assay, using radioactively labelled partner DNA strands, quantified the density of functional DNA strands attached to the carbon nanotubes. The diversity of potential applications for these DNA-modified carbon-nanotube platforms is exemplified here by the successful use of a DNA-modified single-walled carbon-nanotube mat as an electrode for the specific detection of metal ions.

Well-defined cholesterol polymers with pH-controlled membrane switching activity.

Cholesterol has been used as an effective component of therapeutic delivery systems because of its ability to cross cellular membranes. Considering this, well-defined copolymers of methacrylic acid and cholesteryl methacrylate, poly(methacrylic acid-co-cholesteryl methacrylate) P(MAA-co-CMA), were generated as potential delivery system components for pH-controlled intracellular delivery of therapeutics. Statistical copolymers with varying cholesterol contents (2, 4, and 8 mol %) were synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization. Dynamic light scattering (DLS) analysis showed that the hydrodynamic diameters of the copolymers in aqueous solutions ranged from 5 ± 0.3 to 7 ± 0.4 nm for the copolymers having 2 and 4 mol % CMA and 8 ± 1.1 to 13 ± 1.9 nm for the copolymer having 8 mol % CMA with increasing pH (pH 4.5-7.4). Atomic force microscopy (AFM) analysis revealed that the copolymer having 8 mol % CMA formed supramolecular assemblies while the copolymers having 2 and 4 mol % CMA existed as unimers in aqueous solution. The pH-responsive behavior of the copolymers was investigated via UV-visible spectroscopy revealing phase transitions at pH 3.9 for 2 mol % CMA, pH 4.7 for 4 mol % CMA, and pH 5.4 for 8 mol % CMA. Lipid bilayers and liposomes as models for cellular membranes were generated to probe their interactions with the synthesized copolymers. The interactions were determined in a pH-dependent manner (at pH 5.0 and 7.4) using surface plasmon resonance (SPR) spectroscopy and liposome leakage assay. Both the SPR analyses and liposome leakage assays indicated that the copolymer containing 2 mol % CMA displayed the greatest polymer-lipid interactions at pH 5.0, presenting the highest binding ability to the lipid bilayer surfaces, and also demonstrating the highest membrane destabilization activity. CellTiter-Blue assay showed that the copolymers did not affect the cell viability up to 30 µM over a period of 72 h.

Exploiting zinc oxide re-emission to fabricate periodic arrays.

The synthesis of hexagonal ring-shaped structures of zinc oxide using nanosphere lithography and metal/metal oxide sputtering is demonstrated. This synthesis exploits the surface re-emission of zinc oxide to deposit material in regions lying out of the line-of-sight of the sputtering source. These rings can nucleate the hydrothermal growth of zinc oxide crystals. Control over the growth could be exercised by varying growth solution concentration or temperature or by applying an external potential.

Molecular recognition and supramolecular self-assembly of a genetically engineered gold binding peptide on Au{111}.

The understanding of biomineralization and realization of biology-inspired materials technologies depends on understanding the nature of the chemical and physical interactions between proteins and biominerals or synthetically made inorganic materials. Recently, combinatorial genetic techniques permit the isolation of peptides recognizing specific inorganic materials that are used as molecular building blocks for novel applications. Little is known about the molecular structure of these peptides and the specific recognition mechanisms onto their counterpart inorganic surfaces. Here, we report high-resolution atomic force microscopy (AFM), molecular simulation (MS), and geometrical docking studies that detail the formation of an ordered supramolecular self-assembly of a genetically engineered gold binding peptide, 3rGBP(1) ([MHGKTQATSGTIQS](3)), correlating with the symmetry of the Au{111} surface lattice. Using simulated annealing molecular dynamics (SA/MD) studies based on nuclear magnetic resonance (NMR), we confirmed the intrinsic disorder of 3rGBP(1) and identified putative Au docking sites where surface-exposed side chains align with both the <110> and <211> Miller indices of the Au lattice. Our results provide fundamental insight for an atomistic understanding of peptide/solid interfaces and the intrinsic disorder that is inherent in some of these peptide sequences. Analogous to the well-established atomically controlled thin-film heterostructure formation on semiconductor substrates, the basis of today's microelectronics, the fundamental observations of peptide-solid interactions here may well form the basis of peptide-based hybrid molecular technologies of the future.

Structure and properties of redox active self-assembled monolayers formed from norbornylogous bridges.

Three different length rigid norbornylogous bridges with terminal ferrocene moieties were synthesized. Pure self-assembled monolayers (SAMs) of the norbornylogous bridges and SAMs with the bridges diluted using either hydroxyl-terminated or methyl-terminated diluents were formed for each length of norbornylogous bridge. The SAMs were imaged with scanning tunneling micrsocopy (STM) and the electrochemical properties were investigated. It was found that SAMs composed of only norbornylogous bridges were crystalline-like, while in mixed SAMs, where the norbornylogous bridge was diluted, the ferrocene stood above the surface of the diluent because of the rigidity of the norbornylogous bridges and were homogeneously distributed across the surface. Further, the rate of electron transfer of the norbornylogous bridges was observed to be similar to an alkanethiol-derived ferrocene whose construct was designed to be as close as possible to that of the norbornylogous bridge. Finally, the rate of electron transfer for the norbornylogous bridges in a diluted SAM was slower with a hydroxyl-terminated diluent than with a methyl-terminated diluent.

Synthesis of versatile thiol-reactive polymer scaffolds via RAFT polymerization.

Well-defined polymer scaffolds convertible to (multi)functional polymer structures via selective and efficient modifications potentially provide an easy, versatile, and useful approach for a wide variety of applications. Considering this, a homopolymer scaffold, poly(pyridyldisulfide ethylmethacrylate) (poly(PDSM)), having pendant groups selectively reactive with thiols, was synthesized by reversible addition fragmentation chain transfer (RAFT) polymerization. Soluble polymers with controlled molecular weights and narrow PDIs were generated efficiently. The versatility of the scaffold to generate random co- and ter-polymers combining multiple functionalities with controlled-composition was shown by separate and simultaneous conjugation of different mercapto-compounds, including a tripeptide in one-step. Conversion of water-insoluble scaffold to peptide-containing water-soluble copolymers was observed to yield nanometer-size particles with narrow polydispersity. The overall results suggest that the well-defined PDSM homopolymer scaffold generated via RAFT polymerization can be a versatile building block for generation of new structures having potential for drug delivery applications via a straightforward synthetic approach.

Nanocapacitive circuit elements.

"Natural" lithography was used to prepare arrays of nanoscale capacitors on silicon. The capacitance was verified by a novel technique based on the interaction of a charged substrate with the electron beam of a scanning electron microscope. The "nanocapacitors" possessed a capacitance of approximately 1 x 10(-16) F and were observed to hold charge for over an hour. Our results indicate that fabricating nanostructures using natural lithography may provide a viable alternative for future nanoelectronic devices.

Temperature-responsive self-assembled monolayers of oligo(ethylene glycol): control of biomolecular recognition.

Self-assembled monolayers (SAMs) of oligo(ethylene glycol) (OEG)-tethered molecules on gold are important for various biorelevant applications ranging from biomaterials to bioanalytical devices, where surface resistance to nonspecific protein adsorption is needed. Incorporation of a stimuli-responsive character to the OEG SAMs enables the creation of nonfouling surfaces with switchable functionality. Here we present an OEG-derived structure that is highly responsive to temperature changes in the vicinity of the physiological temperature, 37 degrees C. The temperature-responsive solution behavior of this new compound was demonstrated by UV-vis and nuclear magnetic resonance spectroscopy. Its chemisorption onto gold(111), and the retention of responsive behavior after chemisorption have been demonstrated by surface plasmon resonance (SPR), X-ray photoelectron spectroscopy (XPS), and atomic force and scanning tunneling microscopy. The OEG-derived SAMs have been shown to reversibly switch the wettability of the surface, as determined by contact angle measurements. More importantly, SPR and AFM studies showed that the OEG SAMs can be utilized to control the affinity binding of streptavidin to the biotin-tethered surface in a temperature-dependent manner while still offering the nonspecific protein-resistance to the surface.

Ethynylbenzene monolayers on gold: a metal-molecule binding motif derived from a hydrocarbon.

Exposure of a Au(111) surface to ethynylbenzene in solution leads to the formation of a bound monolayer. A chemisorption process occurs to give a stable layer consisting of oxygen-containing hydrocarbon species. Ethynylbenzene itself does not oxidize under the deposition conditions indicating that the gold surface facilitates the oxidation process. Calculations show that ethynylbenzene and its oxidation products phenylacetic acid and phenyloxirane have positive binding energies to the gold surface. 1,4-Diethynylbenzene also binds to Au(111) and anchors gold nanoparticles deposited from solution to form dense, semiregular arrays.

Self-organization of a discotic coordination complex bearing orthogonal discotic ligands.

A new coordination complex that incorporates discotic ligands oriented orthogonally to a central discotic unit has been synthesized, and images of self-organized structures on highly oriented pyrolytic graphite have been obtained. Scanning tunneling microscopy images show that the combination of the orthogonally disposed discotic components results in significantly different self-organized structures compared to those of the ligand, which contains a single discotic unit.