Nanoprecipitation-Enhanced Sensitivity in Enzymatic Nanofluidic Biosensors.

Single nanochannels show unique transport properties due to nanoconfinement. It has been demonstrated that at submillimolar concentrations of divalent cations, a nanoprecipitation reaction can occur in nanochannels. Although several reports have shown, described, and modeled the nanoprecipitation process, no further advantages have been taken from this phenomenon. Here, we show that the nanoprecipitation reaction can be incorporated into enzyme-modified nanochannels to enhance the performance of small-molecule biosensors via in situ amplification reactions. Contrary to the working principle of previous enzymatic nanofluidic biosensors, the nanofluidic biosensor described in this work operates on the basis of concerted functions: pH-shifting enzymatic activity and nanoprecipitation. We show that the simple addition of Ca2+ and Mg2+ ions in the working analyte solution containing urea can lower the detection limit from the nanometer to the subnanometer regime and modulate the dynamic linear range. This approach enables the implementation of more sensitive real-time nanofluidic detection methods without increasing the complexity of the nanofluidic platform or the sensing approach. We envision that the integration of concerted functions in nanofluidic architectures will play a key role in expanding the use of these nanoscale devices for analytical purposes.

Polyelectrolyte-multivalent molecule complexes: physicochemical properties and applications.

The complexation of polyelectrolytes with other oppositely charged structures gives rise to a great variety of functional materials with potential applications in a wide spectrum of technological fields. Depending on the assembly conditions, polyelectrolyte complexes can acquire different macroscopic configurations such as dense precipitates, nanosized colloids and liquid coacervates. In the past 50 years, much progress has been achieved to understand the principles behind the phase separation induced by the interaction of two oppositely charged polyelectrolytes in aqueous solutions, especially for symmetric systems (systems in which both polyions have similar molecular weight and concentration). However, in recent years, the complexation of polyelectrolytes with alternative building blocks such as small charged molecules (multivalent inorganic species, oligopeptides, and oligoamines, among others) has gained attention in different areas. In this review, we discuss the physicochemical characteristics of the complexes formed by polyelectrolytes and multivalent small molecules, putting a special emphasis on their similarities with the well-known polycation-polyanion complexes. In addition, we analyze the potential of these complexes to act as versatile functional platforms in various technological fields, such as biomedicine and advanced materials engineering.

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.

Insulin Delivery from Glucose-Responsive, Self-Assembled, Polyamine Nanoparticles: Smart "Sense-and-Treat" Nanocarriers Made Easy.

Polyamine-salt aggregates (PSA) are biomimetic soft materials that have attracted great attention due to their straightforward fabrication methods, high drug-loading efficiencies, and attractive properties for pH-triggered release. Herein, a simple and fast multicomponent self-assembly process was used to construct cross-linked poly(allylamine hydrochloride)/phosphate PSAs (hydrodynamic diameter of 360 nm) containing glucose oxidase enzyme, as a glucose-responsive element, and human recombinant insulin, as a therapeutic agent for the treatment of diabetes mellitus (GI-PSA). The addition of increasing glucose concentrations promotes the release of insulin due to the disassembly of the GI-PSAs triggered by the catalytic in situ formation of gluconic acid. Under normoglycemia, the GI-PSA integrity remained intact for at least 24 h, whereas hyperglycemic conditions resulted in 100 % cargo release after 4 h of glucose addition. This entirely supramolecular strategy presents great potential for the construction of smart glucose-responsive delivery nanocarriers.

Structural analysis of a natural apolipoprotein A-I variant (L60R) associated with amyloidosis.

The reason that determines the pathological deposition of human apolipoprotein A-I variants inducing organ failure has been under research since the early description of natural mutations in patients. To shed light into the events associated with protein aggregation, we studied the structural perturbations that may occur in the natural variant that shows a substitution of a Leucine by an Arginine in position 60 (L60R). Circular dichroism, intrinsic fluorescence measurements, and proteolysis analysis indicated that L60R was more unstable, more sensitive to cleavage and the N-terminus was more disorganized than the protein with the native sequence (Wt). A higher tendency to aggregate was also detected when L60R was incubated at physiological pH. In addition, the small structural rearrangement observed for the freshly folded variant led to the release of tumor necrosis factor-α and interleukin-1ß from a model of macrophages. However, the mutant preserved both its dimeric conformation and its lipid-binding capacity. Our results strongly suggest that the chronic disease may be a consequence of the native conformation loss which elicits the release of protein conformations that could be either cytotoxic or precursors of amyloid conformations.

A study of the complex interaction between poly allylamine hydrochloride and negatively charged poly(N-isopropylacrylamide-co-methacrylic acid) microgels.

Negatively charged poly(N-isopropylacrylamide-co-methacrylic acid) (P(NIPAm-co-MAA)) microgels undergo size changes in response to changes in temperature and pH. Complexation of these microgels with positively charged polyelectrolytes can greatly affect their physical properties and their capacity for encapsulating active molecules. Here we study the interaction between (P(NIPAm-co-MAA)) microgels and a model positively charged polyelectrolyte, poly allylamine hydrochloride (PAH), with different molecular weights. Experiments were conducted at temperatures below and above the lower critical solution temperature (LCST) of the microgel (30-32 °C), at 20 and 40 °C, respectively, and for PAH at molecular weights of 15, 50, and 140 kDa. Below the LCST, dynamic light scattering and zeta potential measurements with molecular simulation show that for the 15 kDa PAH there is preferential accumulation of PAH inside the microgel, whereas for the higher molecular weight PAH, the polyelectrolyte deposits mainly on the microgel surface. Above the LCST, PAH is preferentially located on the surface of the microgels for all molecular weights studied as a result of charge segregation in the hydrogels. Confocal scanning laser microscopy and flow cytometry were used to quantify rhodamine labelled PAH associated with the microgel. Isothermal titration calorimetry studies give insight into the thermodynamics of the interaction of PAH with the hydrogels, and how this interaction is affected by the molecular weight of PAH. Finally, microgels with encapsulated doxorubicin were exposed to PAH, revealing that the drug is displaced from the microgel by the PAH chains.

Redox-active polyamine-salt aggregates as multistimuli-responsive soft nanoparticles.

Polyamine-salt aggregates have become promising soft materials in nanotechnology due to their easy preparation process and pH-responsiveness. Here, we report the use of hexacyanoferrate(ii) and hexacyanoferrate(iii) as electroactive crosslinking agents for the formation of nanometer-sized redox-active polyamine-redox-salt aggregates (rPSA) in bulk suspension. This nanoplatform can be selectively assembled or disassembled under different stimuli such as redox environment, pH and ionic strength. By changing the charge of the building blocks, external triggers allow switching the system between two phase states: aggregate-free solution or colloidal rPSA dispersion. The stimuli-activated modulation of the assembly/disassembly processes opens a path to exploit rPSA in technologies based on smart nanomaterials.

Practical use of polymer brushes in sustainable energy applications: interfacial nanoarchitectonics for high-efficiency devices.

The discovery and development of novel approaches, materials and manufacturing processes in the field of energy are compelling increasing recognition as a major challenge for contemporary societies. The performance and lifetime of energy devices are critically dependent on nanoscale interfacial phenomena. From the viewpoint of materials design, the improvement of current technologies inevitably relies on gaining control over the complex interface between dissimilar materials. In this sense, interfacial nanoarchitectonics with polymer brushes has seen growing interest due to its potential to overcome many of the limitations of energy storage and conversion devices. Polymer brushes offer a broad variety of resources to manipulate interfacial properties and gain molecular control over the synergistic combination of materials. Many recent examples show that the rational integration of polymer brushes in hybrid nanoarchitectures greatly improves the performance of energy devices in terms of power density, lifetime and stability. Seen in this light, polymer brushes provide a new perspective from which to consider the development of hybrid materials and devices with improved functionalities. The aim of this review is therefore to focus on what polymer brush-based solutions can offer and to show how the practical use of surface-grafted polymer layers can improve the performance and efficiency of fuel cells, lithium-ion batteries, organic radical batteries, supercapacitors, photoelectrochemical cells and photovoltaic devices.

Polyamine Colloids Cross-Linked with Phosphate Ions: Towards Understanding the Solution Phase Behavior.

Ionically crosslinked poly(allylamine)/phosphate (PAH/Pi) colloids consist of self-assembled nanostructures stabilized by supramolecular interactions. Under physiological conditions, these interactions should be present at high ionic strength and only in a narrow pH window to be effective as drug delivery agents. In this work we study the effect of the pH and ionic strength in the chemical behaviour of inorganic phosphate (Pi), poly(allylamine hydrochloride) (PAH) and their mixture in aqueous solution (PAH-Pi). By combination of experimental measurements and a theoretical model, we demonstrate that the driving force that leads to the formation of colloids is the electrostatic pairing between the positively charged amino groups in PAH and negatively charged HPO42- ions. Increasing the ionic strength of the system by addition of KCl weakens the PAH-Pi interactions and narrows the pH stability window from 4 to 1.8 pH units. In addition, a fully reversible system was obtained in which the colloids assemble and disassemble by changing the pH between 6.8 and 7.1 at high ionic strength, making them suitable for use as pH-responsive nanocarriers.

Continuous assembly of supramolecular polyamine-phosphate networks on surfaces: preparation and permeability properties of nanofilms.

Supramolecular self-assembly of molecular building blocks represents a powerful "nanoarchitectonic" tool to create new functional materials with molecular-level feature control. Here, we propose a simple method to create tunable phosphate/polyamine-based films on surfaces by successive assembly of poly(allylamine hydrochloride) (PAH)/phosphate anions (Pi) supramolecular networks. The growth of the films showed a great linearity and regularity with the number of steps. The coating thickness can be easily modulated by the bulk concentration of PAH and the deposition cycles. The PAH/Pi networks showed chemical stability between pH 4 and 10. The transport properties of the surface assemblies formed from different deposition cycles were evaluated electrochemically by using different redox probes in aqueous solution. The results revealed that either highly permeable films or efficient anion transport selectivity can be created by simply varying the concentration of PAH. This experimental evidence indicates that this new strategy of supramolecular self-assembly can be useful for the rational construction of single polyelectrolyte nanoarchitectures with multiple functionalities.

Short communication: Activity of nisin, lipid bilayer fragments and cationic nisin-lipid nanoparticles against multidrug-resistant Staphylococcus spp. isolated from bovine mastitis.

Staphylococci are the main etiological agents of bovine mastitis. Bacteriocins and nanoparticles have emerged as promising alternatives for the future development of antimicrobial agents. This study evaluated the activity of the bacteriocin nisin and bicelles of the synthetic cationic lipid dioctadecyldimethylammonium bromide, alone and in combination, against multidrug-resistant Staphylococcus spp. strains isolated from bovine mastitis. In summary, cationic nisin/dioctadecyldimethylammonium bromide nanoparticles are shown to be a promising alternative for the control of mastitis caused by multidrug-resistant Staphylococcus spp.

Highly Sensitive Biosensing with Solid-State Nanopores Displaying Enzymatically Reconfigurable Rectification Properties.

Molecular design of biosensors based on enzymatic processes taking place in nanofluidic elements is receiving increasing attention by the scientific community. In this work, we describe the construction of novel ultrasensitive enzymatic nanopore biosensors employing "reactive signal amplifiers" as key elements coupled to the transduction mechanism. The proposed framework offers innovative design concepts not only to amplify the detected ionic signal and develop ultrasensitive nanopore-based sensors but also to construct nanofluidic diodes displaying specific chemo-reversible rectification properties. The integrated approach is demonstrated by electrostatically assembling poly(allylamine) on the anionic pore walls followed by the assembly of urease. We show that the cationic weak polyelectrolyte acts as a "reactive signal amplifier" in the presence of local pH changes induced by the enzymatic reaction. These bioinduced variations in proton concentration ultimately alter the protonation degree of the polyamine resulting in amplifiable, controlled, and reproducible changes in the surface charge of the pore walls, and consequently on the generated ionic signals. The "iontronic" response of the as-obtained devices is fully reversible, and nanopores are reused and assayed with different urea concentrations, thus ensuring reliable design. The limit of detection (LOD) was 1 nM. To the best of our knowledge, this value is the lowest LOD reported to date for enzymatic urea detection. In this context, we envision that this approach based on the use of "reactive signal amplifiers" into solid-state nanochannels will provide new alternatives for the molecular design of highly sensitive nanopore biosensors as well as (bio)chemically addressable nanofluidic elements.

Layer-by-Layer Assembled Microgels Can Combine Conflicting Properties: Switchable Stiffness and Wettability without Affecting Permeability.

Responsive interfacial architectures of practical interest commonly require the combination of conflicting properties in terms of their demand upon material structure. Switchable stiffness, wettability, and permeability, key features for tissue engineering applications, are in fact known to be exclusively interdependent. Here, we present a nanoarchitectonic approach that decouples these divergent properties by the use of thermoresponsive microgels as building blocks for the construction of three-dimensional arrays of interconnected pores. Layer-by-layer assembled poly( N-isopropylacrylamide- co-methacrylic acid) microgel films were found to exhibit an increase in hydrophobicity, stiffness, and adhesion properties upon switching the temperature from below to above the lower critical solution temperature, whereas the permeability of redox probes through the film remained unchanged. Our findings indicate that the switch in hydrophilicity and nanomechanical properties undergone by the microgels does not compromise the porosity of the film, thus allowing the free diffusion of redox probes through the polymer-free volume of the submicrometer pores. This novel approach for decoupling conflicting properties provides a strategic route for creating tailorable scaffolds with unforeseen functionalities.

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.

Systemic availability of guanidinoacetate affects GABAA receptor function and seizure threshold in GAMT deficient mice.

Deficiency of guanidinoacetate methyltransferase (GAMT) causes creatine depletion and guanidinoacetate accumulation in brain with the latter deemed to be responsible for the severe seizure disorder seen in affected patients. We studied electrical brain activity and GABAA mediated mechanisms of B6J.Cg-Gamt(tm1Isb) mice. Electrocorticographic (ECoG) monitoring of pharmacological treatments with ornithine (5 % in drinking water for 5-18 days) and/or Picrotoxin (PTX) (a GABAA receptor antagonist) (1.5 mg/kg, I.P.) in Gamt(MUT) and Gamt(WT) groups [n = 3, mean age (SEM) = 6.9 (0.2) weeks]. Mice were fitted with two frontal and two parietal epidural electrodes under ketamine/xylazine anesthesia. Baseline and test recordings were performed for determination of seizure activity over a 2 h period. The ECoG baseline of Gamt(MUT) exhibited an abnormal monotonous cortical rhythm (7-8 Hz) with little variability during awake and sleep states compared to wild type recordings. Ornithine treatment and also PTX administration led to a relative normalization of the Gamt(MUT) ECoG phenotype. Gamt(WT) on PTX exhibited electro-behavioral seizures, whereas the Gamt(MUT) did not have PTX induced seizures at the same PTX dose. Gamt(MUT) treated with both ornithine and PTX did not show electro-behavioral seizures while ornithine elevated the PTX seizure threshold of Gamt(MUT) mice even further. These data demonstrate: (1) that there is expression of electrical seizure activity in this Gamt-deficient transgenic mouse strain, and (2) that the systemic availability of guanidinoacetate affects GABAA receptor function and seizure thresholds. These findings are directly and clinically relevant for patients with a creatine-deficiency syndrome due to genetic defects in GAMT and provide a rational basis for a combined ornithine/picrotoxin therapeutic intervention.

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.

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.

Trypanosoma cruzi: effects of azadirachtin and ecdysone on the dynamic development in Rhodnius prolixus larvae.

The effects of azadirachtin and ecdysone on the Trypanosoma cruzi population in the Rhodnius prolixus gut were investigated. T. cruzi were rarely found in the gut compartments of azadirachtin-treated larvae. High parasite numbers were observed in the stomach of the control and ecdysone groups until 10 days after treatment and in the small intestine and rectum until 25 days after treatment. High percentages of round forms developed in the stomachs of all groups, whereas azadirachtin blocked the development of protozoan intermediate forms. This effect was counteracted by ecdysone therapy. In the small intestine and rectum, epimastigotes predominated for all groups, but more of their intermediates developed in the control and ecdysone groups. Azadirachtin supported the development of round forms and their intermediates into trypomastigotes. In the rectum, trypomastigotes did not develop in the azadirachtin group and developed much later after ecdysone therapy. The parallel between the effects of azadirachtin and ecdysone on the host and parasite development is discussed on the basis of the present results because ecdysone appears to act directly or indirectly in determining the synchronic development of T. cruzi forms from round to epimastigotes, but not metacyclic trypomastigotes, in the invertebrate vector.

Highly sensitive acetylcholine biosensing via chemical amplification of enzymatic processes in nanochannels.

Acetylcholinesterase-modified nanochannels are proposed as reliable and reproducible nanofluidic sensors for highly sensitive detection of acetylcholine. The operation mechanism relies on the use of weak polyelectrolytes as "chemical amplifiers" that adjust/reconfigure the nanochannel surface charge in the presence of local pH changes induced by the enzymatic reaction. Experimental results show that the presence of acetylcholine can be transduced into measurable ionic signals with a low limit of detection.