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

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

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 invesigated 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

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

Formation and surface biomolecular interactions of PEG grafted monolayers using SPR, XPS and AFM

Surface analytical tools can be used to characterize the formation and biointeractions of polyethylene glycols (PEG) grafted monolayers adsorbed or tethered to surfaces. Complementary atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and surface plasmon resonance (SPR) analysis were performed to determine the chemisorption behavior of these polymers to metallic surfaces. The data were then applied to qualify the interactions of modified polymer surfaces with proteins observed in situ by SPR

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