Selection of synthetic proteins to modulate the human frataxin function.

Frataxin is a kinetic activator of the mitochondrial supercomplex for iron-sulfur cluster assembly. Low frataxin expression or a decrease in its functionality results in Friedreich's Ataxia (FRDA). With the aim of creating new molecular tools to study this metabolic pathway, and ultimately, to explore new therapeutic strategies, we have investigated the possibility of obtaining small proteins exhibiting a high affinity for frataxin. In this study, we applied the ribosome display approach, using human frataxin as the target. We focused on Affi_224, one of the proteins that we were able to select after five rounds of selection. We have studied the interaction between both proteins and discussed some applications of this specific molecular tutor, concerning the modulation of the supercomplex activity. Affi_224 and frataxin showed a KD value in the nanomolar range, as judged by surface plasmon resonance analysis. Most likely, it binds to the frataxin acidic ridge, as suggested by the analysis of chemical shift perturbations (nuclear magnetic resonance) and computational simulations. Affi_224 was able to increase Cys NFS1 desulfurase activation exerted by the FRDA frataxin variant G130V. Importantly, Affi_224 interacts with frataxin in a human cellular model. Our results suggest quaternary addition may be a new tool to modulate frataxin function in vivo. Nevertheless, more functional experiments under physiological conditions should be carried out to evaluate Affi_224 effectiveness in FRDA cell models.

Unexpected enhancement of pH-stability in Au3+/Ag+ loaded H-bonded layer-by-layer thin films.

In this work, a polymeric film was synthesized through a layer-by-layer (LBL) self-assembly technique using polyacrylic acid (PAA) and polyethylene oxide (PEO), resulting in the formation of a hydrogen-bonded LBL film. The formation of these films was evaluated by PMIRRAS and QCM-D. The synergy of these techniques allowed the understanding of the mechanism of formation of the film by showing the H-bonding formation and film growth. Au and Ag metal ions were successfully incorporated into the films, as corroborated by the combination of the information obtained by XRR and PMIRRAS. The films were exposed to increasing pH, showing a pronounced improvement in stability in films loaded with Au ions, extending the stability from pH 4 to 10. This behavior allows the use of this system in a wider range of applications, including the possibility of working in biological conditions. On the other hand, films loaded with Ag disintegrated at pH above 4. At acidic pH (below 3), these films released the Ag ions, which may be useful for the preparation of antibacterial stimuli-responsive nanomaterials. In both cases, the films were adequate to produce metal nanoparticles by metal loading and in situ reduction.

Enzymes hosted in redox-active ionically cross-linked polyelectrolyte networks enable more efficient biofuel cells.

Redox mediators are pivotal players in the electron transfer process between enzymes and electrodes. We present an alternative approach for redox mediation based on branched polyethyleneimine (BPEI) modified with an osmium complex. This redox polyelectrolyte is crosslinked with phosphate to produce colloidal particles with a diameter of ca. 1 µm, which, combined with glucose oxidase (GOx), can form electroactive assemblies through either layer by layer assembly (LbL) or one-pot drop-casting (OPDC). The addition of NaCl to these colloidal systems induces the formation of films that otherwise poorly grow, presenting an outstanding catalytic current. The system was tested as a bioanode delivering a power output of 148 µW per nmol of mediator. These results are explained in terms of the interactions of the ions with the polyelectrolyte and represent a new route for the development of bioelectrochemical devices involving redox mediators and enzymes.

Efficient decolorization of recalcitrant dyes at neutral/alkaline pH by a new bacterial laccase-mediator system.

Laccases and laccase-mediator systems (LMS) are versatile catalysts that can oxidize a broad range of substrates coupled to the sole reduction of dioxygen to water. They possess many biotechnological applications in paper, textile, and food industries, bioethanol production, organic synthesis, detection and degradation of pollutants, and biofuel cell development. In particular, bacterial laccases are getting relevance due to their activity in a wide range of pH and temperature and their robustness under harsh conditions. However, the enzyme and the redox mediator's availability and costs limit their large-scale commercial use. Here we demonstrate that ß-(10-phenothiazyl)-propionic acid can be used as an efficient and low-cost redox mediator for decolorizing synthetic dyes by the recombinant laccase SilA from Streptomyces ipomoeae produced in E. coli. This new LMS can decolorize more than 80% indigo carmine and malachite green in 1 h at pH = 8.0 and 2 h in tap water (pH = 6.8). Furthermore, it decolorized more than 40% of anthraquinone dye remazol brilliant blue R and 80% of azo dye xylidine ponceau in 5 h at 50 °C, pH 8.0. It supported at least 3 decolorization cycles without losing activity, representing an attractive candidate for a cost-effective and environmentally friendly LMS functional at neutral to alkaline pH.

The effect of ionic strength and phosphate ions on the construction of redox polyelectrolyte-enzyme self-assemblies.

Layer by layer assembly of polyelectrolytes with proteins is a convenient tool for the development of functional biomaterials. Most of the studies presented in the literature are based on the electrostatic interaction between components of opposite charges, limiting the assembly possibilities. However, this process can be tuned by modifying the environment where the main constituents are dissolved. In this work, the electron transfer behavior between an electroactive polyelectrolyte (polyallylamine derivatized with an osmium complex) and a redox enzyme (glucose oxidase) is studied by assembling them in the presence of phosphate ions at different ionic strengths. Our results show that the environment from which the assembly is constructed has a significant effect on the electrochemical response. Notably, the polyelectrolyte dissolved in the presence of phosphate at high ionic strength presents a globular structure which is preserved after adsorption with substantial effects on the buildup of the multilayer system, improving the electron transfer process through the film.

Highly-organized stacked multilayers via layer-by-layer assembly of lipid-like surfactants and polyelectrolytes. Stratified supramolecular structures for (bio)electrochemical nanoarchitectonics.

Supramolecular self-assembly is of paramount importance for the development of novel functional materials with molecular-level feature control. In particular, the interest in creating well-defined stratified multilayers through simple methods using readily available building blocks is motivated by a multitude of research activities in the field of "nanoarchitectonics" as well as evolving technological applications. Herein, we report on the facile preparation and application of highly organized stacked multilayers via layer-by-layer assembly of lipid-like surfactants and polyelectrolytes. Polyelectrolyte multilayers with high degree of stratification of the internal structure were constructed through consecutive assembly of polyallylamine and dodecyl phosphate, a lipid-like surfactant that act as a structure-directing agent. We show that multilayers form well-defined lamellar hydrophilic/hydrophobic domains oriented parallel to the substrate. More important, X-ray reflectivity characterization conclusively revealed the presence of Bragg peaks up to fourth order, evidencing the highly stratified structure of the multilayer. Additionally, hydrophobic lamellar domains were used as hosts for ferrocene in order to create an electrochemically active film displaying spatially-addressed redox units. Stacked multilayers were then assembled integrating redox-tagged polyallylamine and glucose oxidase into the stratified hydrophilic domains. Bioelectrocatalysis and "redox wiring" in the presence of glucose was demonstrated to occur inside the stratified multilayer.

Surfactants as mesogenic agents in layer-by-layer assembled polyelectrolyte/surfactant multilayers: nanoarchitectured "soft" thin films displaying a tailored mesostructure.

Interfacial supramolecular architectures displaying mesoscale organized components are of fundamental importance for developing materials with novel or optimized properties. Nevertheless, engineering the multilayer assembly of different building blocks onto a surface and exerting control over the internal mesostructure of the resulting film is still a challenging task in materials science. In the present work we demonstrate that the integration of surfactants (as mesogenic agents) into layer-by-layer (LbL) assembled polyelectrolyte multilayers offers a straightforward approach to control the internal film organization at the mesoscale level. The mesostructure of films constituted of hexadecyltrimethylammonium bromide, CTAB, and polyacrylic acid, PAA (of different molecular weights), was characterized as a function of the number of assembled layers. Structural characterization of the multilayered films by grazing-incidence small-angle X-ray scattering (GISAXS), showed the formation of mesostructured composite polyelectrolyte assemblies. Interestingly, the (PAA/CTA)n assemblies prepared with low PAA molecular weight presented different mesostructural regimes which were dependent on the number of assembled layers: a lamellar mesophase for the first bilayers, and a hexagonal circular mesophase for n ≥ 7. This interesting observation was explained in terms of the strong interaction between the substrate and the first layers leading to a particular mesophase. As the film increases its thickness, the prevalence of this strong interaction decreases and the supramolecular architecture exhibits a "bulk" mesophase. Finally, we demonstrated that the molecular weight of the polyelectrolyte has a considerable impact on the meso-organization for the (PAA/CTA)n assemblies. We consider that these studies open a path to new rational methodologies to construct "nanoarchitectured" polyelectrolyte multilayers.

Numerical Simulation of the Diffusion Processes in Nanoelectrode Arrays Using an Axial Neighbor Symmetry Approximation.

Nanoelectrode arrays have introduced a complete new battery of devices with fascinating electrocatalytic, sensitivity, and selectivity properties. To understand and predict the electrochemical response of these arrays, a theoretical framework is needed. Cyclic voltammetry is a well-fitted experimental technique to understand the undergoing diffusion and kinetics processes. Previous works describing microelectrode arrays have exploited the interelectrode distance to simulate its behavior as the summation of individual electrodes. This approach becomes limited when the size of the electrodes decreases to the nanometer scale due to their strong radial effect with the consequent overlapping of the diffusional fields. In this work, we present a computational model able to simulate the electrochemical behavior of arrays working either as the summation of individual electrodes or being affected by the overlapping of the diffusional fields without previous considerations. Our computational model relays in dividing a regular electrode array in cells. In each of them, there is a central electrode surrounded by neighbor electrodes; these neighbor electrodes are transformed in a ring maintaining the same active electrode area than the summation of the closest neighbor electrodes. Using this axial neighbor symmetry approximation, the problem acquires a cylindrical symmetry, being applicable to any diffusion pattern. The model is validated against micro- and nanoelectrode arrays showing its ability to predict their behavior and therefore to be used as a designing tool.

Effect of gold nanoparticles on the structure and electron-transfer characteristics of glucose oxidase redox polyelectrolyte-surfactant complexes.

Efficient electrical communication between redox proteins and electrodes is a critical issue in the operation and development of amperometric biosensors. The present study explores the advantages of a nanostructured redox-active polyelectrolyte-surfactant complex containing [Os(bpy)2Clpy](2+) (bpy=2,2'-bipyridine, py= pyridine) as the redox centers and gold nanoparticles (AuNPs) as nanodomains for boosting the electron-transfer propagation throughout the assembled film in the presence of glucose oxidase (GOx). Film structure was characterized by grazing-incidence small-angle X-ray scattering (GISAXS) and atomic force microscopy (AFM), GOx incorporation was followed by surface plasmon resonance (SPR) and quartz-crystal microbalance with dissipation (QCM-D), whereas Raman spectroelectrochemistry and electrochemical studies confirmed the ability of the entrapped gold nanoparticles to enhance the electron-transfer processes between the enzyme and the electrode surface. Our results show that nanocomposite films exhibit five-fold increase in current response to glucose compared with analogous supramolecular AuNP-free films. The introduction of colloidal gold promotes drastic mesostructural changes in the film, which in turn leads to a rigid, amorphous interfacial architecture where nanoparticles, redox centers, and GOx remain in close proximity, thus improving the electron-transfer process.

Hydrophobic interactions leading to a complex interplay between bioelectrocatalytic properties and multilayer meso-organization in layer-by-layer assemblies.

The present study explores the development of mesostructured bioelectrochemical interfaces with accurate compositional and topological control of the supramolecular architecture through the layer-by-layer assembly of ternary systems based on poly(allylamine) containing an osmium polypyridyl complex (OsPA), an anionic surfactant, sodium dodecyl sulfate (SDS) or sodium octodecyl sulfate (ODS), and glucose oxidase (GOx). We show that the introduction of the anionic surfactant allows a sensitive increase of the polyelectrolyte and the enzyme uptake at pH 7.0, enhancing its catalytic behavior in the presence of glucose as compared to the surfactant-free system (OsPA/GOx)n constructed at the same pH. Structural characterization of the multilayer films was performed by means of grazing-incidence small-angle X-ray scattering (GISAXS), which showed the formation of mesostructured domains within the composite assemblies. Experimental results indicate that the balance between ionic and hydrophobic interactions plays a leading role not only in the construction of the self-assembled system but also in the functional properties of the bioactive interface. The structure of the ternary multilayered films depends largely on the length of the alkyl chain of the surfactant. We show that surfactants incorporated into the film also play a role as chemical entities capable of tuning the hydrophobicity of the whole assembly. In this way, the deliberate introduction of short-range hydrophobic forces was exploited as an additional variable to manipulate the adsorption and coverage of protein during each assembly step. However, the integration of long-chain surfactants may lead to the formation of very well-organized interfacial architectures with poor electron transfer properties. This, in turn, leads to a complex trade-off between enzyme coverage and redox wiring that is governed by the meso-organization and the hydrophobic characteristics of the multilayer assembly.

Quantification of Enzymatic Biofilm Removal Using the Sauerbrey Equation: Application to the Case of Pseudomonas protegens.

A methodology for the quantitative analysis of enzymatic removal of biofilms (BF) was developed, based on a quartz crystal microbalance (QCM) under stationary conditions. This was applied to the case of Pseudomonas protegens (PP) BFs, through a series of five enzymes, whose removal activity was screened using the presented methodology. The procedure is based on the following: when BFs can be modeled as rigid materials, QCM can be used as a balance under stationary conditions for determining the BFs mass reduction by enzymatic removal. For considering a BF as a rigid model, energy dissipation effects, associated with viscoelastic properties of the BF, must be negligible. Hence, a QCM system with detection of dissipation (referred to as QCM with dissipation) was used for evaluating the energy losses, which, in fact, resulted in negligible energy losses in the case of dehydrated PP BFs, validating the application of the Sauerbrey equation for the change of mass calculations. The stationary methodology reduces operating times and simplifies data analysis in comparison to dynamic approaches based on flow setups, which requires the incorporation of dissipation effects due to the liquid media. By carrying out QCM, glycosidase-type enzymes showed BF removal higher than 80% at enzyme concentration 50 ppm, reaching removal over 90% in the cases of amylase and cellulase/xylanase enzymes. The highest removal percentage produced a reduction from about 15 to 1 µg in the BF mass. Amylase enzyme was tested from below 50 to 1 ppm, reaching around 60% of removal at 1 ppm. The obtained results were supported by other instrumental techniques such as Raman spectroscopy, attenuated total reflection Fourier transform infrared spectroscopy, atomic force microscopy, high performance anion exchange chromatography, thermogravimetric analysis, and differential scanning calorimetry. The removal quantifications obtained with QCM were compared with those obtained by well-established screening techniques (UV-vis spectrophotometry using crystal violet and agar diffusion test). The proposed methodology expands the possibility of using a quartz microbalance to perform enzymatic activity screening.

Electron transfer properties of dual self-assembled architectures based on specific recognition and electrostatic driving forces: its application to control substrate inhibition in horseradish peroxidase-based sensors.

This work describes the synergistic combination of ionic self-assembly and recognition-directed assembly with the aim of creating highly functional bioelectrochemical interfaces compatible with the supramolecular design of a wide variety of biosensing platforms. A recently synthesized glycopolyelectrolyte constituted of polyallylamine bearing redox-active osmium complexes and glycosidic residues (lactose) is used to create a self-assembled structure with sodium dodecylsulfate. In turn, this supramolecular thin films bearing redox-active and biorecognizable carbohydrate units enable the facile assembly of functional lectins as well as the subsequent docking and "wiring" of glycoenzymes, like horseradish peroxidase (HRP) (an elusive enzyme to immobilize via noncovalent interactions). The assembly of this system was followed by quartz crystal microbalance and grazing-incidence small-angle X-ray scattering (GISAXS) studies confirming that spin-coated ionically self-assembled films exhibit mesostructured architectures according to the formation of self-organized lamellar structures. In-depth characterization of the electrocatalytic properties of the biosupramacromolecular assemblies confirmed the ability of this kind of interfacial architecture to efficiently mediate electron transfer processes between the glycoenzyme and the electrode surface. For instance, our experimental electrochemical evidence clearly shows that tailor-made interfacial configurations of the ionic self-assemblies can prevent the inhibition of the glycoenzyme (typically observed in HRP) leading to bioelectrocatalytic currents up to 0.1 mA cm(-2). The presence of carbohydrate moieties in the ionic domains promotes the biorecognition-driven assembly of lectins adding a new dimension to the capabilities of ionic self-assembly.

Electrochemical Actuation of a DyP Peroxidase: A Facile Method for Drastic Improvement of the Catalytic Performance.

Dye decolorizing peroxidases (DyP) have attracted interest for applications such as dye-containing wastewater remediation and biomass processing. So far, efforts to improve operational pH ranges, activities, and stabilities have focused on site-directed mutagenesis and directed evolution strategies. Here, we show that the performance of the DyP from Bacillus subtilis can be drastically boosted without the need for complex molecular biology procedures by simply activating the enzyme electrochemically in the absence of externally added H2O2. Under these conditions, the enzyme shows specific activities toward a variety of chemically different substrates that are significantly higher than in its canonical operation. Moreover, it presents much broader pH activity profiles with the maxima shifted toward neutral to alkaline. We also show that the enzyme can be successfully immobilized on biocompatible electrodes. When actuated electrochemically, the enzymatic electrodes have two orders of magnitude higher turnover numbers than with the standard H2O2-dependent operation and preserve about 30% of the initial electrocatalytic activity after 5 days of operation-storage cycles.

Phase Behavior and Electrochemical Properties of Highly Asymmetric Redox Coacervates.

This work reports the phase behavior and electrochemical properties of liquid coacervates made of ferricyanide and poly(ethylenimine). In contrast to the typical polyanion/polycation pairs used in liquid coacervates, the ferricyanide/poly(ethylenimine) system is highly asymmetric because poly(ethylenimine) has approximately 170 charges per molecule, while ferricyanide has only 3. Two types of phase diagrams were measured and fitted with a theoretical model. In the first type of diagram, the stability of the coacervate was studied in the plane given by the concentration of poly(ethylenimine) versus the concentration of ferricyanide for a fixed concentration of added monovalent salt (NaCl). The second type of diagram involved the plane given by the concentration of poly(ethylenimine) vs the concentration of the added monovalent salt for a fixed poly(ethyleneimine)/ferricyanide ratio. Interestingly, these phase diagrams displayed qualitative similarities to those of symmetric polyanion/polycation systems, suggesting that coacervates formed by a polyelectrolyte and a small multivalent ion can be treated as a specific case of polyelectrolyte coacervate. The characterization of the electrochemical properties of the coacervate revealed that the addition of monovalent salt greatly enhances charge transport, presumably by breaking ion pairs between ferricyanide and poly(ethylenimine). This finding highlights the significant influence of added salt on the transport properties of coacervates. This study provides the first comprehensive characterization of the phase behavior and transport properties of asymmetric coacervates and places these results within the broader context of the better-known symmetric polyelectrolyte coacervates.

Development of a Redox-Polymer-Based Electrochemical Glucose Biosensor Suitable for Integration in Microfluidic 3D Cell Culture Systems.

The inclusion of online, in situ biosensors in microfluidic cell cultures is important to monitor and characterize a physiologically mimicking environment. This work presents the performance of second-generation electrochemical enzymatic biosensors to detect glucose in cell culture media. Glutaraldehyde and ethylene glycol diglycidyl ether (EGDGE) were tested as cross-linkers to immobilize glucose oxidase and an osmium-modified redox polymer on the surface of carbon electrodes. Tests employing screen printed electrodes showed adequate performance in a Roswell Park Memorial Institute (RPMI-1640) media spiked with fetal bovine serum (FBS). Comparable first-generation sensors were shown to be heavily affected by complex biological media. This difference is explained in terms of the respective charge transfer mechanisms. Under the tested conditions, electron hopping between Os redox centers was less vulnerable than H2O2 diffusion to biofouling by the substances present in the cell culture matrix. By employing pencil leads as electrodes, the incorporation of these electrodes in a polydimethylsiloxane (PDMS) microfluidic channel was achieved simply and at a low cost. Under flow conditions, electrodes fabricated using EGDGE presented the best performance with a limit of detection of 0.5 mM, a linear range up to 10 mM, and a sensitivity of 4.69 µA mM-1 cm-2.

Recognition-driven layer-by-layer construction of multiprotein assemblies on surfaces: a biomolecular toolkit for building up chemoresponsive bioelectrochemical interfaces.

The development of soft bioelectronic interfaces with accurate compositional and topological control of the supramolecular architecture attracts intense interest in the fast-growing field of bioelectronics and biosensing. The present study explores the recognition-driven layer-by-layer assembly of glycoenzymes onto electrode surfaces. The design of the multi-protein interfacial architecture is based on the multivalent supramolecular carbohydrate-lectin interactions between redox glycoproteins and concanavalin A (Con A) derivatives. Specifically, [Os(bpy)(2)Clpy](2+)-tagged Con A (Os-Con A) and native Con A were used to direct the assembly of horseradish peroxidase (HRP) and glucose oxidase (GOx) in a stepwise topologically controlled procedure. In our designed configuration, GOx acts as the biorecognition element to glucose stimulus, while HRP acts as the transducing element. Surface plasmon resonance (SPR) spectroscopy and quartz crystal microbalance with dissipation (QCM-D) results are combined to give a close representation of the protein surface coverage and the content of water in the protein assembly. The characterization is complemented with in situ atomic force microscopy (AFM) to give a topographical description of the layers assemblage. Electrochemical (EC) techniques were used to characterize the functional features of the spontaneously self-assembled biohybrid architecture, showing that the whole system presents efficient electron transfer and mass transport processes being able to transform micromolar glucose concentration into electrical information. In this way the combination of the electroactive and nonelectroactive Con A provides an efficient strategy to control the position and composition of the protein layers via recognition-driven processes, which defines its sensitivity toward glucose. Furthermore, the incorporation of dextran as a permeable interlayer able to bind Con A promotes the physical separation of the biochemical and transducing processes, thus enhancing the magnitude of the bioelectrochemical signal. We consider that these results are relevant for the nanoconstruction of functional biointerfaces provided that our experimental evidence reveals the possibility of locally addressing recognition, transduction and amplification elements in interfacial ensembles via LbL recognition-driven processes.

Electrochemical sensing platform based on polyelectrolyte-surfactant supramolecular assemblies incorporating carbon nanotubes.

The characterization and application of a polyelectrolyte-surfactant supramolecular assembly formed by poly(allylamine) and dodecyl sulfate (PA-DS) on a screen-printed graphite electrode for the preparation of electrochemical sensing platforms are presented. The system was characterized by X-ray reflectometry (XRR) and grazing-incidence small-angle X-ray scattering (GISAXS) and tested with four benchmark electrochemical probes undergoing different electron-transfer mechanisms on carbon: ferrocyanide, hexaammineruthenium, ascorbic acid, and dopamine. The polyelectrolyte acts as a scaffold favoring the incorporation of the ferrocyanide, an ion oppositely charged to poly(allylamine). Also, its ability to incorporate carbon nanotubes (CNT) is presented. The composite material PA-DS-CNT is able to electrocatalyze the oxidation of dopamine, allowing its detection at micromolar levels in the presence of 100 times higher concentrations of ascorbate and it is shown to be stable, while XRR and GISAXS results confirm a lamellar structure with well-defined domains, not perturbed by the presence of the CNT. The dispersion is easily prepared in aqueous solution and could facilitate the processing of the CNT with an efficient loading and yielding a more robust carbon-based material for sensing applications.

Mass and charge transport in highly mesostructured polyelectrolyte/electroactive-surfactant multilayer films.

HYPOTHESIS: Dimensionally stable electroactive films displaying spatially addressed redox sites is still a challenging goal due to gel-like structure. Polyelectrolyte and surfactants can yield highly mesostructured films using simple buildup strategies as layer-by-layer. The use of redox modified surfactants is expected to introduce order and an electroactive response in thin films. EXPERIMENTS: The assembly of polyacrylic acid and different combinations of redox-modified and unmodified hexadecyltrimethylammonium bromide yields highly structured and electroactive thin films. The growth, viscoelastic properties, mass, and electron transport of these films were studied by combining electrochemical and quartz crystal balance with dissipation experiments. FINDINGS: Our results show that the films are highly rigid and poorly hydrated. The mass and charge transport reveal that the ingress (egress) of the counter ions during the electrochemical oxidation (reduction) is accompanied with a small amount of water, which is close to their hydration sphere. Thus, the generated mesostructured films present an efficient charge transport with negligible changes in their structures during the electron transfer process. The control over the meso-organization and its stability represents a promising tool in the construction of devices where the vectorial transfer of electrons, or ions, is required.

Redox-active concanavalin A: synthesis, characterization, and recognition-driven assembly of interfacial architectures for bioelectronic applications.

The convergence of chemistry, biology, and materials science has paved the way to the emergence of hybrid nanobuilding blocks that incorporate the highly selective recognition properties of biomolecules, with the tailorable functional capabilities of inorganic molecules. In this work, we describe for the first time the decoration of concanavalin A (Con A), a protein with the ability to recognize sugars and form glycoconjugates, with Os(II) redox-active complexes. This strategy enabled the construction of electroactive biosupramolecular materials whose redox potentials could be easily modulated through the facile molecular modification of the electroactive inorganic complexes. Small-angle X-ray scattering (SAXS), steady-state fluorescence, surface plasmon resonance (SPR) spectroscopy, matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS), and differential-pulsed (DPV) and cyclic voltammetry (CV) were used to characterize the structural and functional features of the synthesized biohybrid building blocks as well as their respective supramolecular assemblies built up on gold electrodes. By harnessing the electroactive and carbohydrate-recognition properties of these tailor-made biohybrid building blocks, we were able to integrate glucose oxidase (GOx) onto gold electrodes via sugar-lectin interactions. The redox activity of the Os-modified Con A interlayer allowed the electronic connection between the multilayered GOx assemblies and the metal electrode as evidenced by the well-defined bioelectrocatalytic response exhibited by the biomolecular assemblies in the presence of the glucose in solution. We consider that this approach based on the spontaneous formation of redox-active biosupramolecular assemblies driven by recognition processes can be of practical relevance for the facile design of biosensors, as well as for the construction of new multifunctional bioelectrochemical systems.

Supramolecular assembly of glucose oxidase on concanavalin A--modified gold electrodes.

There is a growing quest for the construction of functional supramolecular architectures to efficiently translate (bio)chemical events into easily measurable signals. This interest originates from its inherent scientific relevance as well as from their potential applications in the ever-flourishing areas of bioelectronics and biosensing. Herein, we describe the immobilization of glycoproteins onto electrode surfaces based on recognition-mediated supramolecular processes. Quartz crystal microbalance with dissipation (QCM-D), surface plasmon resonance (SPR) spectroscopy, and electrochemical (EC) measurements were used to characterize the structural and functional features of these bio-supramolecular systems. Carbohydrate-lectin interactions were successfully used to build up stable assemblies of glucose oxidase (GOx) layers mediated by the recognition properties of concanavalin A supramolecular architectures. The catalytic response of GOx indicates that the whole population of enzymes incorporated in the supramolecular architecture is fully active. Even though lectin-carbohydrate interactions are rather weak, the multivalency effects prevailing in the supramolecular assembly confer remarkable stability to the interfacial architecture, thus preventing the release of the enzyme from the surface even with high glucose (ligand) concentrations. This approach represents a simple and straightforward route to locally address functional glycoproteins at interfaces. In this context, we consider that the versatility of a supramolecular assembly using biological interactions could open up new ways of envisioning or to generate new ideas for the future development of highly efficient bioelectronic platforms.