M. Gabriella Santonicola

Switching Transport through Nanopores with pH-Responsive Polymer Brushes for Controlled Ion Permeability

Several nanoporous platforms were functionalized with pH-responsive poly(methacrylic acid) (PMAA) brushes using surface-initiated atom transfer radical polymerization (SI-ATRP). The growth of the PMAA brush and its pH-responsive behavior from the nanoporous platforms were confirmed by scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, and atomic force microscopy (AFM). The swelling behavior of the pH-responsive PMAA brushes grafted only from the nanopore walls was investigated by AFM in aqueous liquid environment with pH values of 4 and 8. AFM images displayed open nanopores at pH 4 and closed ones at pH 8, which rationalizes their use as gating platforms. Ion conductivity across the nanopores was investigated with current-voltage measurements at various pH values. Enhanced higher resistance across the nanopores was observed in a neutral polymer brush state (lower pH values) and lower resistance when the brush was charged (higher pH values). By adding a fluorescent dye in an environment of pH 4 or pH 8 at one side of the PMAA-brush functionalized nanopore array chips, diffusion across the nanopores was followed. These experiments displayed faster diffusion rates of the fluorescent molecules at pH 4 (PMAA neutral state, open pores) and slower diffusion at pH 8 (PMAA charged state, closed pores) showing the potential of this technology toward nanoscale valve applications.

Reversible ph-controlled switching of poly(methacrylic acid) grafts for functional biointerfaces

Responsive polymeric brushes of poly(methacrylic acid) (PMAA) were grafted from silicon surfaces using controlled surface-initiated atom-transfer radical polymerization (SI-ATRP). The growth kinetics of PMAA was investigated with respect to the composition of the ATRP medium by grafting the polymer in mixtures of water and methanol with different ratios. The dissociation behavior of the polymer layers was characterized by FTIR titration after incubating the polymer-grafted substrates in PBS buffer solutions with different pH values. PMAA layers show a strong pH-dependent behavior with an effective pK(a) of the bulk polymer brush of 6.5 ± 0.2, which is independent of the polymer brush thickness and methanol content of the ATRP grafting medium. The pH-induced swelling and collapse of the grafted polymer layers were quantified in real time by in situ ellipsometry in liquid environment. Switching between polymer conformations at pH values of 4 and 8 is rapid and reversible, and it is characterized by swelling factors (maximum thickness/minimum thickness) that increase with decreasing the methanol content of the SI-ATRP medium.

In situ monitoring of the catalytic activity of cytochrome c oxidase in a biomimetic architecture

Cytochrome c oxidase (CcO) from Paracoccus denitrificans was immobilized in a strict orientation via a his-tag attached to subunit I on a gold film and reconstituted in situ into a protein-tethered bilayer lipid membrane. In this orientation, the cytochrome c (cyt c) binding site is directed away from the electrode pointing to the outer side of the protein-tethered bilayer lipid membrane architecture. The CcO can thus be activated by cyt c under aerobic conditions. Catalytic activity was monitored by impedance spectroscopy, as well as cyclic voltammetry. Cathodic and anodic currents of the CcO with cyt c added to the bulk solution were shown to increase under aerobic compared to anaerobic conditions. Catalytic activity was considered in terms of repeated electrochemical oxidation/reduction of the CcO/cyt c complex in the presence of oxygen. The communication of cyt c bound to the CcO with the electrode is discussed in terms of a hopping mechanism through the redox sites of the enzyme. Simulations supporting this hypothesis are included.

Surface-grafted zwitterionic polymers as platforms for functional supported phospholipid membranes

Polymer brushes grafted from surfaces using controlled polymerization techniques, most notably surface-initiated atom-transfer radical polymerization (SI-ATRP), provide robust and reproducible platforms with precise control of surface properties. These platforms are especially useful in biologically oriented applications involving the confinement of membrane proteins onto solid supports, including screening of pharmaceuticals and biosensing. Here we investigate a tunable zwitterion-based polymeric interface that can guide the assembly of neutral lipid membranes with high mechanical stability and reproducibility on various synthetic materials. By controlling the polymer architecture using ATRP, we show that phospholipid membranes can be made to self-assemble on thin layers of charge-balanced poly(sulfobetaine methacrylate) from fusion of DOPC vesicles under physiological conditions. The self-assembly kinetics and functionality of the polymer-supported lipid membranes are investigated using various surface sensitive techniques, including surface plasmon resonance, fluorescence microscopy, and atomic force microscopy. The growth of zwitterionic polymer layers with controlled length and grafting density allows for modulation of the adhesion of the lipid bilayers to surfaces, thus offering unique advantages for the design and synthesis of bioactive surfaces

Binding of alkyl polyglucoside surfactants to bacteriorhodopsin and its relation to protein stability.

The binding of alkyl polyglucoside surfactants to the integral membrane protein bacteriorhodopsin (BR) and the formation of protein-surfactant complexes are investigated by sedimentation equilibrium via analytical ultracentrifugation and by small-angle neutron scattering (SANS). Contrast variation techniques in SANS enable measurement of the composition of the protein-surfactant complexes and determination of the thickness of the surfactant shell bound to the protein. The results indicate that alkyl polyglucosides can bind to BR as single surfactant layers or as a thicker shell. The thickness of the surfactant shell increases with increasing surfactant tail length, and it is generally unrelated to the aggregation number of the micelles even for a small and predominantly hydrophobic membrane protein such as BR. The aggregation numbers determined by sedimentation equilibrium methods match those measured by SANS, which also allows reconstruction of the shape of the protein-detergent complex. When the surfactant is present as a single layer, the BR loses activity, as measured by absorption spectroscopy, more quickly than it does when the surfactant forms a thicker shell.

Surface-enhanced Raman spectroscopy characterisation of functionalised multi-walled carbon nanotubes

Multi-walled nanotube (MWNT) functionalisation was investigated by surface-enhanced Raman spectroscopy (SERS). The MWNTs were deposited as dilute dispersions on SERS-active substrates. We used nano-structured gold surfaces with various morphologies for our measurements. The surface enhancement effect was used to amplify the Raman signal from functional molecules bound to the nanotube walls. The recorded spectral features allowed for discrimination between the differently functionalised MWNTs. Although the present study is limited to a few examples, our measurements indicate the higher specificity obtained by the SERS approach and its possible use for a systematic study of functionalisation effects on MWNT structures.

A supramolecular two-photon-active hydrogel platform for direct bioconjugation under near-infrared radiation

A supramolecular hydrogel assembled from partially methacrylated polyethyleneimine (PEI) and with direct photopatterning capabilities at near-infrared (NIR) wavelengths is presented. The chemically modified branched PEI macromolecules were characterized by FTIR and NMR spectroscopy to quantify the degree of methacrylation. A highly hydrophilic polymer network with a water content up to 95% was prepared. The hydrogel microstructure in an aqueous solution was characterized using confocal laser scanning microscopy (CLSM), which revealed a porous network with large interconnected cavities. The photo-sensitive PEIMA hydrogel was activated by two-photon laser irradiation and micropatterns formed at its interface when probes with free carboxylic acid or hydroxyl groups were present in solution. Direct patterning of the hydrogel matrix with different biomolecules, and without additional photoinitiators, is demonstrated in two-photon microscopy experiments.

Nanomaterial-based biosensors for a real-time detection of biological damage by UV light.

In this work, the design and fabrication of a miniaturized and light-weight biosensor that can be used to monitor the biological effects of hostile ultraviolet radiation in earth and space are presented. The biosensor is generated by embedding a sensitive element to UV radiation, DNA, in a hybrid carbon-based nanomaterial. In particular, we present results on the fabrication and characterization of hybrid nanostructured films containing graphene nanoplatelets (GNPs) and double-stranded DNA for the in situ and real-time detection of UV radiation damaging effects from the changes of the film electrical properties induced by exposure to UV-C radiation. The biosensor is realized by the deposition of the sensitive unit GNP/DNA on a supporting substrate made of flexible polymers or glass.

Supramolecular polycationic hydrogels with high swelling capacity prepared by partial methacrylation of polyethyleneimine

Methacrylation of branched polyethyleneimine (PEI) was performed to generate modified polycationic macromolecules that self-assemble in water into highly swollen supramolecular hydrogels with tunable hydrophilicity and microstructure. The properties of the supramolecular hydrogels, in terms of swelling and porosity, were controlled during synthesis by the extent of methacrylation of the starting PEI macromolecules. The methacrylation reaction was conducted under several conditions, and the methacrylated PEI (PEI-MA) molecules were investigated by FTIR and NMR to relate the reaction parameters to the amount of methacrylate moieties on the modified polymer. The hydrogel morphology and its dynamic nature arising from the non-covalent interactions among the PEI-MA macromolecules were characterized by small-angle X-ray scattering (SAXS) and fluorescence microscopy at different temperatures, which enabled visualization of the large interconnected microcavities of the supramolecular gel network.

Steady-shear rheological properties of graphene-reinforced epoxy resin for manufacturing of aerospace composite films

The aim of this work is to analyze the steady-shear rheological behavior and the absolute viscosity of epoxy matrix reinforced with graphene nanoplatelets (xGnP) before cure. Three different grades of xGnP (grades C, M and H) were dispersed homogenously at different weight percentages (wt%) into the epoxy matrix, ranging from 0.5 to 5 wt%. It is found that nanocomposite fluids with xGnP-C exhibit a Newtonian behavior at shear rate in the range 0.1–100 s−1, conversely, nanocomposite fluids with xGnP of grade M and H exhibit a shear-thinning behavior with the increase of nanoplatelet loading. Results from this analysis indicate how the steady shear rheological properties of the nano-reinforced polymer fluids depend on the geometrical characteristics of the graphene nanoplatelets.