Disruption of diphenylalanine assembly by a Boc-modified variant.

Peptide-based biomaterials are key to the future of diagnostics and therapy, promoting applications such as tissue scaffolds and drug delivery vehicles. To realise the full potential of the peptide systems, control and optimisation of material properties are essential. Here we investigated the co-assembly of the minimal amyloid motif peptide, diphenylalanine (FF), and its tert-butoxycarbonyl (Boc)-modified derivative. Using Atomic Force Microscopy, we demonstrated that the co-assembled fibers are less rigid and show a curvier morphology. We propose that the Boc-modification of FF disrupts the hydrogen bond packing of adjacent N-termini, as supported by Fourier transform infrared and fluorescence spectroscopic data. Such rationally modified co-assemblies offer chemical functionality for after-assembly modification and controllable surface properties for tissue engineering scaffolds, along with tunable morphological vs. mechanical properties.

Near-field Raman spectroscopy of biological nanomaterials by in situ laser-induced synthesis of tip-enhanced Raman spectroscopy tips.

We report a new approach in tip-enhanced Raman spectroscopy (TERS) in which TERS-active tips with enhancement factors of ∼10(-5)× can be rapidly (1-3 min) produced in situ by laser-induced synthesis of silver nanoparticles at the tip apex. The technique minimizes the risks of tip contamination and damage during handling and provides in situ feedback control, which allows the prediction of the tip performance. We show that TERS tips produced by this technique enable the measurement of spatially resolved TERS spectra of self-assembled peptide nanotubes with a spatial resolution of ∼20 nm.

Investigations of the supramolecular structure of individual diphenylalanine nano- and microtubes by polarized Raman microspectroscopy.

Polarized Raman microspectroscopy and atomic force microscopy were used to obtain quantitative information regarding the molecular structure of individual diphenylalanine (FF) nano- and microtubes. The frequencies of the Raman spectral bands corresponding to the amide I (1690 cm(-1)) and amide III (1249 cm(-1)) indicated that the FF-molecules interact by hydrogen bonding at the N-H and not at the C═O sites. The calculated mean orientation angles of the principal axes of the Raman tensors (PARTs) obtained from the polarized Raman spectral measurements were 41 ± 4° for the amide I and 59 ± 5° for amide III. On the basis of the orientation of the PART for the amide I mode, it was found that the C═O bond is oriented at an angle of 8 ± 4° to the tube axis. These values did not vary significantly with the diameter of the tubes (range 400-1700 nm) and were in agreement with the molecular structure proposed previously for larger crystalline specimens.

The structure and formation of hydrogen-bonded molecular networks on Au(111) surfaces revealed by scanning tunnelling and torsional-tapping atomic force microscopy.

A comprehensive scanning probe microscopy study has been carried out to characterise 3,4,9,10-Perylenetetracarboxylic diimide (PTCDI)-melamine hydrogen-bonded networks deposited on Au(111)-surfaces. Both scanning tunnelling and atomic force microscopy were utilized. Such complementary analysis revealed a multilayered structure of the networks on the Au(111)-surface as opposed to a widely reported monolayer structure. Details of the network formation mechanism are presented. We have also demonstrated that despite the apparent network stability in ambient conditions it is unstable in aqueous solutions of pH 4.5 and 7.1.

Interactions between signal-transducing proteins measured by atomic force microscopy.

Atomic force microscopy (AFM) has been used to study the specific interactions between the signal-transducing proteins mammalian phospholipase D1 (PLD1), phospholipase C-gamma1 (PLC-gamma1), and Munc-18-1. To record the forces between them, the Phox homology (PX) domain of PLD1, the Src homology (SH3) domain of PLC-gamma1, and Munc-18-1 were fused with glutathione S-transferase (GST) and immobilized onto reduced glutathione (GSH)-tethered surfaces. In order to enhance the recognition efficiency and avoid undesirable complications, both AFM tips and substrates were first modified with dendrons of two different sizes. Under the employed conditions, the probability of observing an unbinding event increased, most force-distance curves showed the single rupture events, and the unbinding forces were 51 +/- 2 pN for PX-(Munc-18-1) and 42 +/- 2 pN for PX-SH3. To investigate dynamics of these biomolecular interactions, we measured the loading rate dependence of the unbinding forces. The unbinding forces increased linearly with the logarithm of the loading rate, indicating the presence of a single potential barrier in the dissociation energy landscape. The measured off-rate constants (k(off)) at 15 degrees C were 10(-3.4 +/- 0.3) s(-1) for PX-(Munc-18-1) and 10(-1.7 +/- 0.1) s(-1) for PX-SH3. Further, we elucidated the influence of free SH3 and Munc-18-1 on the specific PX-(Munc-18-1) and PX-SH3 interaction, respectively.

Enhanced distance-dependent fluorescence quenching using size tuneable core shell silica nanoparticles.

Silica nanoparticles (SNPs) have been used as favoured platforms for sensor, drug delivery and biological imaging applications, due to their ease of synthesis, size-control and bespoke physico-chemical properties. In this study, we have developed a protocol for the synthesis of size-tuneable SNPs, with diameters ranging from 20 nm to 500 nm, through the optimisation of experimental components required for nanoparticle synthesis. This protocol was also used to prepare fluorescent SNPs, via covalent linkages of fluorophores, to the nanoparticle matrix using 3-aminopropyltriethoxysilane (APTES). This enabled the fabrication of ratiometric, fluorescent, pH-sensitive nanosensors (75 nm diameter) composed SNPs covalently linked to two pH-sensitive fluorescent dyes Oregon Green (OG) and 5(6)-carboxyfluorescein (FAM) and a reference fluorescent dye 5-(6)-carboxytetramethylrhodamine (TAMRA), extending the dynamic range of measurement from pH 3.5 to 7.5. In addition, size-tuneable, core-shell SNPs, covalently linked to a fluorescent TAMRA core were synthesised to investigate distance-dependant fluorescence quenching between TAMRA and black hole quencher 2 (BHQ2®) using nanometre-sized silica shells as physical spacers. The results showed a significant fluorescence quenching could be observed over greater distances than that reported for the classical distance-dependent molecular fluorescence quenching techniques, e.g. the Förster (fluorescence) resonance energy transfer (FRET). The methods and protocols we have detailed in this manuscript will provide the basis for the reproducible production of size tunable SNPs, which will find broad utility in the development of sensors for biological applications.

Real-time measurement of the intracellular pH of yeast cells during glucose metabolism using ratiometric fluorescent nanosensors.

Intracellular pH is a key parameter that influences many biochemical and metabolic pathways that can also be used as an indirect marker to monitor metabolic and intracellular processes. Herein, we utilise ratiometric fluorescent pH-sensitive nanosensors with an extended dynamic pH range to measure the intracellular pH of yeast (Saccharomyces cerevisiae) during glucose metabolism in real-time. Ratiometric fluorescent pH-sensitive nanosensors consisting of a polyacrylamide nanoparticle matrix covalently linked to two pH-sensitive fluorophores, Oregon green (OG) and 5(6)carboxyfluorescein (FAM), and a reference pH-insensitive fluorophore, 5(6)carboxytetramethylrhodamine (TAMRA), were synthesised. Nanosensors were functionalised with acrylamidopropyltrimethyl ammonium hydrochloride (ACTA) to confer a positive charge to the nanoparticle surfaces that facilitated nanosensor delivery to yeast cells, negating the need to use stress inducing techniques. The results showed that under glucose-starved conditions the intracellular pH of yeast population (n ≈ 200) was 4.67 ± 0.15. Upon addition of d-(+)-glucose (10 mM), this pH value decreased to pH 3.86 ± 0.13 over a period of 10 minutes followed by a gradual rise to a maximal pH of 5.21 ± 0.26, 25 minutes after glucose addition. 45 minutes after the addition of glucose, the intracellular pH of yeast cells returned to that of the glucose starved conditions. This study advances our understanding of the interplay between glucose metabolism and pH regulation in yeast cells, and indicates that the intracellular pH homestasis in yeast is highly regulated and demonstrates the utility of nanosensors for real-time intracellular pH measurements.

AFM studies on the role of the protein RdgC in bacterial DNA recombination.

Genetic studies of rdgC in different bacterial systems suggest that it may play a role in replication and recombination. However, the exact function of the corresponding protein, RdgC, is unknown. In this study, we have imaged complexes of RdgC with both linear and supercoiled circular plasmid DNA using atomic force microscopy. We confirm that RdgC does not target any specific sequences in double-stranded DNA, as has been suggested from biochemical data. However, we detect an increased affinity of the protein to DNA ends, and an ability to promote bending of DNA. Similar binding preferences have been reported for enzymes involved in recombination. Protein complexes with supercoiled plasmid DNA further enabled us to study the effect of RdgC on DNA superstructure. At high concentrations of protein we observed promotion of DNA condensation. Recombination is largely enhanced by close contacts of distant regions along the DNA strands, as can occur, for instance, through condensation. Our data thus support a possible function of RdgC as a midwife of recombination.

Direct real-time molecular scale visualisation of the degradation of condensed DNA complexes exposed to DNase I.

The need to protect DNA from in vivo degradation is one of the basic tenets of therapeutic gene delivery and a standard test for any proposed delivery vector. The currently employed in vitro tests, however, presently provide no direct link between the molecular structure of the vector complexes and their success in this role, thus hindering the rational design of successful gene delivery agents. Here we apply atomic force microscopy (AFM) in liquid to visualise at the molecular scale and in real time, the effect of DNase I on generation 4 polyamidoamine dendrimers (G4) complexed with DNA. These complexes are revealed to be dynamic in nature showing a degree of mobility, in some cases revealing the addition and loss of dendrimers to individual complexes. The formation of the G4-DNA complexes is observed to provide a degree of protection to the DNA. This protection is related to the structural morphology of the formed complex, which is itself shown to be dependent on the dendrimer loading and the time allowed for complex formation.

Controlling the Physical Dimensions of Peptide Nanotubes by Supramolecular Polymer Coassembly.

Molecular self-assembly of peptides into ordered nanotubes is highly important for various technological applications. Very short peptide building blocks, as short as dipeptides, can form assemblies with unique mechanical, optical, piezoelectric, and semiconductive properties. Yet, the control over nanotube length in solution has remained challenging, due to the inherent sequential self-assembly mechanism. Here, in line with polymer chemistry paradigms, we applied a supramolecular polymer coassembly methodology to modulate peptide nanotube elongation. Utilizing this approach, we achieved a narrow, controllable nanotube length distribution by adjusting the molecular ratio of the diphenylalanine assembly unit and its end-capped analogue. Kinetic analysis suggested a slower coassembly organization process as compared to the self-assembly dynamics of each of the building blocks separately. This is consistent with a hierarchal arrangement of the peptide moieties within the coassemblies. Mass spectrometry analysis demonstrated the bimolecular composition of the coassembled nanostructures. Moreover, the peptide nanotubes' length distribution, as determined by electron microscopy, was shown to fit a fragmentation kinetics model. Our results reveal a simple and efficient mechanism for the control of nanotube sizes through the coassembly of peptide entities at various ratios, allowing for the desired end-product formation. This dynamic size control offers tools for molecular engineering at the nanoscale exploiting the advantages of molecular coassembly.

Atomic force microscopy study of human amylin (20-29) fibrils.

Here we present atomic force microscopy images of the fibrils formed by human amylin(20-29). This peptide is a fragment of the polypeptide amylin, the major proteinaceous component of amyloid deposits found in cases of type-II diabetes mellitus. Our results demonstrate that the amylin(20-29) peptide fragment forms amyloid-like fibrils that display polymorphic structures. Twisting along the axis of fibrils was often observed in fibrils aged for 6 hours but disappeared in mature fibrils aged for longer time periods.

Spatial confinement of neurite regrowth from dorsal root ganglia within nonporous microconduits.

Tissue engineering is founded on the concept of controlling the behavior of individual cells to stimulate tissue formation. This control is achieved by mimicking signals that manage natural tissue development or repair. These interdependent signals include cytokine delivery, extracellular matrix interactions, and cell-cell communication. Here, we report on the effect of spatial guidance as a signal for nerve tissue regeneration, using a simple in vitro model. We observe the acceleration of neurite extension from rat dorsal root ganglia within micron-scale tubes. Within these hydrogel-filled conduits, neurites were observed to extend more rapidly than when cultured within the hydrogel alone. The spatial cue also induced a change in tissue architecture, with the cabling of cells within the microconduit. The acceleration of neurite extension was found to be independent of conduit diameter within the range of 200 to 635 microm. Finally, our in vitro model enabled quantification of the effect of combining spatial control and localized nerve growth factor delivery.

The effects of additives on the growth and morphology of paracetamol (acetaminophen) crystals.

It is well known that the presence of impurities can dramatically affect the nucleation, morphology, and chemical properties of crystals. Although literature is replete with examples of impurity or additive-induced modifications of crystals, few have examined the interaction of these compounds with distinct growing faces. In this study, we utilize atomic force microscopy (AFM) and scanning electron microscopy (SEM) to investigate the influence of two structurally related additives of paracetamol (acetaminophen) on its crystal morphology. We also probe, in situ, the effects of these additives on the morphology and growth rate of steps on the (0 0 1) face of the crystal. This study, in conjunction with further investigations, aims to establish the specific mechanisms of inhibition of these additives on each face of paracetamol, and provide a means of overcoming the poor compaction behaviour of paracetamol.

Differential scanning calorimetry and scanning thermal microscopy analysis of pharmaceutical materials.

Micro-thermal analysis (microTA) by scanning thermal microscopy is being used increasingly for the analysis of pharmaceutical dosage forms. However, there is currently little evidence to show that microTA data can compare directly with that from the established approach of differential scanning calorimetry (DSC). This work compares DSC and microTA data from an active vitamin B6 analogue, pyridoxal hydrochloride, and two commonly used pharmaceutical excipients, Mannitol and Avicel which are used in its formulation. It is found that microTA provides precise and accurate micro-thermal analytical data with 0.1 K thermal sensitivity, which is comparable to that obtained by DSC measurements of bulk samples. It is also shown that microTA offers the opportunity to study single particles and the interfacial region between particles, data which is currently inaccessible through the DSC technique.

Study of NAP adsorption and assembly on the surface of HOPG.

NAP is an octapeptide that has demonstrated a neuroprotective/therapeutic efficacy at very low concentrations in preclinical studies and in a number of clinical trials. Yet little is known about its structural organization at low concentrations. Here, we have employed atomic force microscopy to investigate NAP peptide assembly on graphite in aqueous media at nanomolar concentration. High spatial resolution scans of NAP assemblies reveal their fine structure with clearly resolved single NAP units. This observation leads us to conclude that NAP molecules do not form complex self-assembled structures at nanomolar concentration when adsorbed on graphite surface.

Bimolecular porous supramolecular networks deposited from solution on layered materials: graphite, boron nitride and molybdenum disulphide.

A two-dimensional porous network formed from perylene tetracarboxylic diimide (PTCDI) and melamine may be deposited from solution on the surfaces of highly oriented pyrolytic graphite (HOPG), hexagonal boron nitride (hBN) and molybdenum disulphide (MoS2). Images acquired using high resolution atomic force microscopy (AFM) operating under ambient conditions have revealed that the network forms extended ordered monolayers (>1 µm(2)) on HOPG and hBN whereas on MoS2 much smaller islands are observed.

Surface mediated L-phenylalanyl-L-phenylalanine assembly into large dendritic structures.

We report a new class of dipeptide dendritic structures fabricated on the surface of mica via spin casting and the conditions required to achieve them. Both their structure and formation mechanism have been investigated in detail using Atomic Force Microscopy (AFM) at the nanometre scale. Formation of nanotubular structures and their further interaction is shown to be a key step in dendritic structure growth. A possible candidate for the primary building block in the nanotubular structure has been identified. The dendritic structures were found to be stable in ambient conditions for several months, however, they transform into needle-like crystals upon exposure to 100% (relative humidity) humid air.

Ferritin-based new magnetic force microscopic probe detecting 10 nm sized magnetic nanoparticles.

A single-molecule ferritin picking-up process was realized with the use of AFM, which was enhanced by employing controlled dendron surface chemistry. The approach enabled the placement of a single ferritin protein molecule at the very end of an AFM tip. When used for magnetic force microscopy (MFM) imaging, the tips were able to detect magnetic interactions of approximately 10 nm sized magnetic nanoparticles. The single ferritin tip also showed the characteristics of a "multifunctional" MFM probe that can sense the magnetic force from magnetic materials as well as detect the biomolecular interaction force with DNAs on the surface. The multifunctional tip enabled us not only to investigate the specific molecular interaction but also to image the magnetic interaction between the probe and the substrate, in addition to allowing the common capability of topographic imaging. Because the protein engineering of ferritin and the supporting coordination and conjugation chemistry are well-established, we envisage that it would be straightforward to extend this approach to the development of various single magnetic particle MFM probes of different compositions and sizes.

Surface-templated fibril growth of peptide fragments from the shaft domain of the adenovirus fibre protein.

Here we present a study of five analogues of a fragment from the shaft domain of the adenovirus fibre protein that readily form fibrils under a range of conditions. Using atomic force microscopy the fibrillisation of these peptides at the liquid/solid interface utilizing ordered crystalline substrates has been investigated. Our results demonstrate that the assembly pathway at the liquid/solid interface enables only the formation of truncated fibrillar structures, which align along the substrate's underlying atomic lattice during growth. Furthermore, that the concentration and volume of solution applied can be used to directly control the density of fibrillar coverage at the surface.