Marta Álvarez

Photoactivatable Caged Cyclic RGD Peptide for Triggering Integrin Binding and Cell Adhesion to Surfaces

We report the synthesis and properties of a photoactivatable caged RGD peptide and its application for phototriggering integrin‐ and cell‐binding to surfaces. We analysed in detail 1) the differences in the integrin‐binding affinity of the caged and uncaged forms by quartz crystal microbalance (QCM) studies, 2) the efficiency and yield of the photolytic uncaging reaction, 3) the biocompatibility of the photolysis by‐products and irradiation conditions, 4) the possibility of site, temporal and density control of integrin‐binding and therefore human cell attachment, and 5) the possibility of in situ generation of cell patterns and cell gradients by controlling the UV exposure. These studies provide a clear picture of the potential and limitations of caged RGD for integrin‐mediated cell adhesion and demonstrate the application of this approach to the control and study of cell interactions and responses.

A facile route for the preparation of azide-terminated polymers. "Clicking" polyelectrolyte brushes on planar surfaces and nanochannels

In this work we describe the facile preparation of azide-terminated polymers by conventional radical polymerization (cRP) using azo initiators bearing azide groups. We show that cRP provides a convenient avenue for the preparation of azide end-functional polymers in a one-step process. The versatility of this chemical methodology was demonstrated by the synthesis of unprecedented azide end group-functionalized sodium polystyrene sulfonate (PSSNa) and poly(2-methacryloyloxyethyl-trimethylammonium chloride) (PMETAC) which were then “clicked” onto alkyne-terminated silicon surfaces and polyethylene terephthalate nanochannels to form polyelectrolyte brush layers. The facile synthesis of the end-functionalized macromolecular building blocks will enable the creation of a wide variety of “clickable” architectures using very simple synthetic tools. We are confident that these results will constitute a key element in the “click” chemistry toolbox and, as such, will have strong implications for the molecular design of interfaces using macromolecular architectures.

Tailoring of Poly(ether ether ketone) Surface Properties via Surface-Initiated Atom Transfer Radical Polymerization

The interfacial properties of commercial poly(ether ether ketone) (PEEK) have been tailored by tethering polymeric brushes to the PEEK surface via surface-initiated atom transfer radical polymerization (SI-ATRP). The immobilization of an ATRP initiator on the PEEK surface was achieved by an unprecedented simple two-step wet chemical method. The keto groups at the PEEK surface were first wet chemically reduced to hydroxyl groups, and then 2-bromoisobutyryl groups were covalently anchored at the PEEK surface as ATRP initiator. SI-ATRP was performed at these functionalized PEEK surfaces with the three different monomers: potassium 3-(methacryloyloxy)propane-1-sulfonate (MPS), monomethoxy-terminated oligo(ethylene glycol)methacrylate (MeOEGMA), and N-isopropylacrylamide (NIPAAm). Atomic force microscopy, scanning electron microscopy, attenuated total reflection infrared spectroscopy, water contact angle measurements, and X-ray photoelectron spectroscopy ascertained the successful grafting of these polymer brushes at the PEEK surface. These brush-modified PEEK surfaces exhibited fully the physiochemical properties of the respective polymer brush: the surface with polyMPS brush showed selective staining by electrostatic interaction, while the polyMeOEGMA-modified surface was biorepellent. The surface modified with polyNIPAAm brush demonstrated a thermally responsive polarity change.

Multiphoton reactive surfaces using ruthenium(II) photocleavable cages.

Photoreactive surfaces derived from a new photocleavable surface modification agent and with photosensitivity in the Vis and IR region are described. A ruthenium(II) caged aminosilane, [Ru(bpy)(2)(PMe(3))(APTS)](PF(6))(2), was synthesized and attached to silica surfaces. Light irradiation removed the cage and generated surface patterns with reactive amine groups. The photosensitivity of this compound under single (460 nm) and two-photon (900) excitation is demonstrated. Functional patterns with site-selective attachment of other molecular species are described.

Modulating Surface Density of Proteins via Caged Surfaces and Controlled Light Exposure

We demonstrate the possibility of tuning the degree of functionalization of a surface using photoactivatable chemistries and controlled light exposure. A photosensitive organosilane with a protected amine terminal group and a tetraethyleneglycol spacer was synthesized. A o-nitrobenzyl cage was used as the photoremovable group to cage the amine functionality. Surfaces with phototunable amine densities were generated by controlled irradiation of silica substrates modified with the photosensitive anchor. Protein layers with different densities could be obtained by successive coupling and assembly steps. Protein surface concentrations were quantified by reflectance interference. Our results demonstrate that the protein density correlates with the photogenerated ligand density. The density control was proved over four coupling steps (biotin, SAv, (BT)tris-NTA, MBP, or GFP), indicating that the interactions between underlying layer and soluble targets are highly specific and the immobilized targets at the four levels maintain their full functionality. Protein micropatterns with a gradient of protein density were also obtained.

Near Field Guided Chemical Nanopatterning

This article demonstrates the possibility of creating well-defined and functional surface chemical nanopatterns using the optical near field of metal nanostructures and a photosensitive organic layer. The intensity distribution of the near field controlled the site and the extent of the photochemical reaction at the surface. The resulting pattern was used to guide the controlled assembly of colloids with a complementary surface functionality onto the substrate. Gold colloids of 20 nm diameter were covalently bound to the activated nanosites and proved the functionality of the suboptical wavelength structures and enabled direct visualization by means of electron microscopy. Our results prove, for the first time, the possibility of using optical near field to perform chemical reactions and assembly at the nanoscale.

Electrochemical rectification by redox-labeled bioconjugates: Molecular building blocks for the construction of biodiodes

In the present work, we describe the properties of a bifunctional redox-labeled bioconjugate at electrode surfaces mediating the electron transfer across the electrode-electrolyte interface. We show that the assembly of ferrocene-labeled streptavidin on biotinylated electrodes results in a reproducible unidirectional current flow in the presence of electron donors in solution. Such rectifying films were built up by spontaneous binding of tetrameric streptavidin molecules to biotin centers immobilized on the electrode surface. Due to the high affinity of biotin to streptavidin, such bifunctional films completely bind any biotinylated compounds. The charge transport between donors in solution and the Au electrode is mediated by the ferrocene moieties, allowing us to develop a molecular rectifier. Our experimental results suggest that such redox-labeled proteins with a high binding capacity constitute a promising alternative to organic compounds used in molecular electronics.

Comparison of Different Supramolecular Architectures for Oligonucleotide Biosensing

This work describes a comparative study between two biosensing platforms that are commonly used to immobilize capture probes. These platforms refer to thiolated and biotinylated oligonucleotide strands chemisorbed on Au surfaces (DNA SAM) and bioconjugated on streptavidin (SA) monolayers (SA SAM), respectively. Both interfacial architectures were studied using surface acoustic wave (SAW) devices and surface plasmon spectroscopy (SPR). Our studies indicated that DNA SAM platforms enable higher densities of surface-confined oligonucleotide probes. However, their hybridization efficiency is lower when compared to that obtained in SA SAM platforms, thus impacting on a lower detection limit, 5 nM. Furthermore, binding of SA molecules to the biotinylated targets, in an attempt to enhance the signal in both platforms, revealed striking differences between both architectures. The SA underlayer used in the SA SAM configuration confers nonfouling characteristics to the interfacial assembly, thus precluding the nonspecific binding of SA onto the surface. The antifouling behavior of the SA DNA platform is an important feature to be considered in the amplification of hybridization events through the bioconjugation of biotinylated targets with streptavidin-based tags.

Molecular architectures for electrocatalytic amplification of oligonucleotide hybridization

In this work, we describe a new platform suitable for electrocatalytic amplification of oligonucleotide hybridization based on the use of supramolecular bioconjugates incorporating ferrocene-labeled streptavidin. Our goals were aimed at designing a biosensing platform which could support highly reproducible and stable electrocatalytic amplification with maximum efficiency. The use of nonlabeled streptavidin as an underlying layer promotes a major improvement on the characteristics of the amplified electrochemical signal. In addition, the electrocatalytic current can be easily amplified by tuning the concentration of electron donor species in solution. Because of the fact that the redox labels are bioconjugated to the DNA strands, increasing the ionic strength does not lead to the loss of redox labels. More importantly, increasing the concentration of donors only involves the magnification of the signal without implying the permeation of donors (thus reducing the efficient electrocatalysis). This approach represents a major improvement on the use of electrocatalytically amplified DNA-sensing platforms, thus overcoming any possible limitation in connection with the reproducibility and reliability of this well-established method.

Anti-fouling characteristics of surface-confined oligonucleotide strands bioconjugated on streptavidin platforms in the presence of nanomaterials.

This work describes our studies on the molecular design of interfacial architectures suitable for DNA sensing which could resist non-specific binding of nanomaterials commonly used as labels for amplifying biorecognition events. We observed that the non-specific binding of bio-nanomaterials to surface-confined oligonucleotide strands is highly dependent on the characteristics of the interfacial architecture. Thiolated double stranded oligonucleotide arrays assembled on Au surfaces evidence significant fouling in the presence of nanoparticles (NPs) at the nanomolar level. The non-specific interaction between the oligonucleotide strands and the nanomaterials can be sensitively minimized by introducing streptavidin (SAv) as an underlayer conjugated to the DNA arrays. The role of the SAv layer was attributed to the significant hydrophilic repulsion between the SAv-modified surface and the nanomaterials in close proximity to the interface, thus conferring outstanding anti-fouling characteristics to the interfacial architecture. These results provide a simple and straightforward strategy to overcome the limitations introduced by the non-specific binding of labels to achieve reliable detection of DNA-based biorecognition events.