Catalase Detection via Membrane-Based Pressure Sensors.

Membrane-based sensors (MePSs) exhibit remarkable precision and sensitivity in detecting pressure changes. MePSs are commonly used to monitor catalytic reactions in solution, generating gas products crucial for signal amplification in bioassays. They also allow for catalyst quantification by indirectly measuring the pressure generated by the gaseous products. This is particularly interesting for detecting enzymes in biofluids associated with disease onset. To enhance the performance of a MePS, various structural factors influence membrane flexibility and response time, ultimately dictating the device's pressure sensitivity. In this study, we fabricated MePSs using polydimethylsiloxane (PDMS) and investigated how structural modifications affect the Young's modulus (E) and residual stress (σ0) of the membranes. These modifications have a direct impact on the sensors' sensitivity to pressure variations, observed as a function of the volume of the chamber (Σ) or of the mechanical properties of the membrane itself (S). MePSs exhibiting the highest sensitivities were then employed to detect catalyst quantities inducing the dismutation of hydrogen peroxide, producing dioxygen as a gaseous product. As a result, a catalase enzyme was successfully detected using these optimized MePSs, achieving a remarkable sensitivity of (22.7 ± 1.2) µm/nM and a limit of detection (LoD) of 396 pM.

Polymeric Membranes Doped with Halloysite Nanotubes Imaged using Proton Microbeam Microscopy.

Polymeric membranes are useful tools for water filtration processes, with their performance strongly dependent on the presence of hydrophilic dopants. In this study, polyaniline (PANI)-capped aluminosilicate (halloysite) nanotubes (HNTs) are dispersed into polyether sulfone (PES), with concentrations ranging from 0.5 to 1.5 wt%, to modify the properties of the PES membrane. Both undoped and HNT-doped PES membranes are investigated in terms of wettability (static and time-dependent contact angle), permeance, mechanical resistance, and morphology (using scanning electron microscopy (SEM)). The higher water permeance observed for the PES membranes incorporating PANI-capped HNTs is, finally, assessed and discussed vis-à-vis the real distribution of HNTs. Indeed, the imaging and characterization in terms of composition, spatial arrangement, and counting of HNTs embedded within the polymeric matrix are demonstrated using non-destructive Micro Particle Induced X-ray Emission (µ-PIXE) and Scanning Transmission Ion Microscopy (STIM) techniques. This approach not only exhibits the unique ability to detect/highlight the distribution of HNTs incorporated throughout the whole thickness of polymer membranes and provide volumetric morphological information consistent with SEM imaging, but also overcomes the limits of the most common analytical techniques exploiting electron probes. These aspects are comprehensively discussed in terms of practical analysis advantages.

Sunset Yellow Confined in Curved Geometry: A Microfluidic Approach.

The behavior of lyotropic chromonic liquid crystals (LCLCs) in confined environments is an interesting research field that still awaits exploration, with multiple key variables to be uncovered and understood. Microfluidics is a highly versatile technique that allows us to confine LCLCs in micrometric spheres. As microscale networks offer distinct interplays between the surface effects, geometric confinement, and viscosity parameters, rich and unique interactions emerging at the LCLC-microfluidic channel interfaces are expected. Here, we report on the behavior of pure and chiral doped nematic Sunset Yellow (SSY) chromonic microdroplets produced through a microfluidic flow-focusing device. The continuous production of SSY microdroplets with controllable size gives the possibility to systematically study their topological textures as the function of their diameters. Indeed, doped SSY microdroplets produced via microfluidics, show topologies that are typical of common chiral thermotropic liquid crystals. Furthermore, few droplets exhibit a peculiar texture never observed for chiral chromonic liquid crystals. Finally, the achieved precise control of the produced LCLC microdroplets is a crucial step for technological applications in biosensing and anticounterfeiting.

A microfluidic method for passive trapping of sperms in microstructures.

Sperm motility is a prerequisite for male fertility. Enhancing the concentration of motile sperms in assisted reproductive technologies - for human and animal reproduction - is typically achieved through aggressive methods such as centrifugation. Here, we propose a passive technique for the amplification of motile sperm concentration, with no externally imposed forces or flows. The technique is based on the disparity between probability rates, for motile cells, of entering and escaping from complex structures. The effectiveness of the technique is demonstrated in microfluidic experiments with microstructured devices, comparing the trapping power in different geometries. In these micro-traps, we observe an enhancement of cells' concentration close to 10, with a contrast between motile and non-motile cells increased by a similar factor. Simulations of suitable interacting model sperms in realistic geometries reproduce quantitatively the experimental results, extend the range of observations and highlight the components that are key to the optimal trap design.

Advancements in modelling human blood brain-barrier on a chip.

The human Blood Brain Barrier (hBBB) is a complex cellular architecture separating the blood from the brain parenchyma. Its integrity and perfect functionality are essential for preventing neurotoxic plasma components and pathogens enter the brain. Although vital for preserving the correct brain activity, the low permeability of hBBB represents a huge impediment to treat mental and neurological disorders or to address brain tumors. Indeed, the vast majority of potential drug treatments are unable to reach the brain crossing the hBBB. On the other hand, hBBB integrity can be damaged or its permeability increase as a result of infections or in presence of neurodegenerative diseases. Currentin vitrosystems andin vivoanimal models used to study the molecular/drug transport mechanism through the hBBB have several intrinsic limitations that are difficult to overcome. In this scenario, Organ-on-Chip (OoC) models based on microfluidic technologies are considered promising innovative platforms that combine the handiness of anin vitromodel with the complexity of a living organ, while reducing time and costs. In this review, we focus on recent advances in OoCs for developing hBBB models, with the aim of providing the reader with a critical overview of the main guidelines to design and manufacture a hBBB-on-chip, whose compartments need to mimic the 'blood side' and 'brain side' of the barrier, to choose the cells types that are both representative and convenient, and to adequately evaluate the barrier integrity, stability, and functionality.

Potential of CO2-laser processing of quartz for fast prototyping of microfluidic reactors and templates for 3D cell assembly over large scale.

Carbon dioxide (CO2)-laser processing of glasses is a versatile maskless writing technique to engrave micro-structures with flexible control on shape and size. In this study, we present the fabrication of hundreds of microns quartz micro-channels and micro-holes by pulsed CO2-laser ablation with a focus on the great potential of the technique in microfluidics and biomedical applications. After discussing the impact of the laser processing parameters on the design process, we illustrate specific applications. First, we demonstrate the use of a serpentine microfluidic reactor prepared by combining CO2-laser ablation and post-ablation wet etching to remove surface features stemming from laser-texturing that are undesirable for channel sealing. Then, cyclic olefin copolymer micro-pillars are fabricated using laser-processed micro-holes as molds with high detail replication. The hundreds of microns conical and square pyramidal shaped pillars are used as templates to drive 3D cell assembly. Human Umbilical Vein Endothelial Cells are found to assemble in a compact and wrapping way around the micro-pillars forming a tight junction network. These applications are interesting for both Lab-on-a-Chip and Organ-on-a-Chip devices.

New perspectives in oral peptide delivery.

Owing to their structural diversity, peptides are a unique source of innovative active ingredients. However, their development has been challenging because of their disadvantageous pharmacokinetic (PK) properties. Over the past decade, many attempts have been made to improve the oral bioavailability of peptide drugs. In this review, we highlight the most recent and promising techniques aimed at the improvement of the oral bioavailability of peptides. The most recent findings will influence future approaches of pharmaceutical companies in the development of new, more efficient, and safer orally delivered peptides.

Analogy between periodic patterns in thin smectic liquid crystal films and the intermediate state of superconductors.

Spontaneous breaking of symmetry in liquid crystal (LC) films often reveals itself as a microscopic pattern of molecular alignment. In a smectic-A LC, the emergence of positional order at the transition from the nematic phase leads to periodic textures that can be used as optical microarrays, templates for soft lithography, and ordering matrices for the organization and manipulation of functional nanoparticles. While both 1d and 2d patterns have been obtained as a function of the LC film thickness and applied fields, the connection has not been made between pattern formation and the peculiar critical behavior of LCs at the nematic-smectic transition, still eluding a comprehensive theoretical explanation. In this article, we demonstrate that an intense bend distortion applied to the LC molecular director while cooling from the nematic phase produces a frustrated smectic phase with depressed transition temperature, and the characteristic 1d periodic texture previously observed in thin films and under applied electric fields. In light of De Gennes' analogy with the normal-superconductor transition of a metal, we identify the 1d texture as the equivalent of the intermediate state in type I superconductors. The bend distortion is analog to the magnetic field in metals and penetrates in the frustrated phase as an array of undercooled nematic domains, periodically intermixed with bend-free smectic-A domains. Our findings provide fundamental evidence for theories of the nematic-smectic transition, highlighting the deep connection between phase frustration and pattern formation, and perspectives on the design of functional smectic microarrays.

An SPR based immunoassay for the sensitive detection of the soluble epithelial marker E-cadherin.

Protein biomarkers are important diagnostic tools for cancer and several other diseases. To be validated in a clinical context, a biomarker should satisfy some requirements including the ability to provide reliable information on a pathological state by measuring its expression levels. In parallel, the development of an approach capable of detecting biomarkers with high sensitivity and specificity would be ideally suited for clinical applications. Here, we performed an immune-based label free assay using Surface Plasmon Resonance (SPR)-based detection of the soluble form of E-cadherin, a cell-cell contact protein that is involved in the maintaining of tissue integrity. With this approach, we obtained a specific and quantitative detection of E-cadherin from a few hundred microliters of serum of breast cancer patients by obtaining a 10-fold enhancement in the detection limit over a traditional colorimetric ELISA.

Highly Sensitive Membrane-Based Pressure Sensors (MePS) for Real-Time Monitoring of Catalytic Reactions.

Functional, flexible, and integrated lab-on-chips, based on elastic membranes, are capable of fine response to external stimuli, so to pave the way for many applications as multiplexed sensors for a wide range of chemical, physical and biomedical processes. Here, we report on the use of elastic thin membranes (TMs), integrated with a reaction chamber, to fabricate a membrane-based pressure sensor (MePS) for reaction monitoring. In particular, the TM becomes the key-element in the design of a highly sensitive MePS capable to monitor gaseous species production in dynamic and temporally fast processes with high resolution and reproducibility. Indeed, we demonstrate the use of a functional MePS integrating a 2 µm thick polydimethylsiloxane TM by monitoring the dioxygen evolution resulting from catalytic hydrogen peroxide dismutation. The operation of the membrane, explained using a diffusion-dominated model, is demonstrated on two similar catalytic systems with catalase-like activity, assembled into polyelectrolyte multilayers capsules. The MePS, tested in a range between 2 and 50 Pa, allows detecting a dioxygen variation of the µmol L-1 s-1 order. Due to their structural features, flexibility of integration, and biocompatibility, the MePSs are amenable of future development within advanced lab-on-chips.

Continuous-Flow Production of Injectable Liposomes via a Microfluidic Approach.

Injectable liposomes are characterized by a suitable size and unique lipid mixtures, which require time-consuming and nonstraightforward production processes. The complexity of the manufacturing methods may affect liposome solubility, the phase transition temperatures of the membranes, the average particle size, and the associated particle size distribution, with a possible impact on the drug encapsulation and release. By leveraging the precise steady-state control over the mixing of miscible liquids and a highly efficient heat transfer, microfluidic technology has proved to be an effective and direct methodology to produce liposomes. This approach results particularly efficient in reducing the number of the sizing steps, when compared to standard industrial methods. Here, Microfluidic Hydrodynamic Focusing chips were produced and used to form liposomes upon tuning experimental parameters such as lipids concentration and Flow-Rate-Ratios (FRRs). Although modelling evidenced the dependence of the laminar flow on the geometric constraints and the FRR conditions, for the specific formulation investigated in this study, the lipids concentration was identified as the primary factor influencing the size of the liposomes and their polydispersity index. This was attributed to a predominance of the bending elasticity modulus over the vesiculation index in the lipid mixture used. Eventually, liposomes of injectable size were produced using microfluidic one-pot synthesis in continuous flow.

Catalytic self-propulsion of supramolecular capsules powered by polyoxometalate cargos.

Multicompartment, spherical microcontainers were engineered through a layer-by-layer polyelectrolyte deposition around a fluorescent core while integrating a ruthenium polyoxometalate (Ru4POM), as molecular motor, vis-à-vis its oxygenic, propeller effect, fuelled upon H2O2 decomposition. The resulting chemomechanical system, with average speeds of up to 25 µm s(-1), is amenable for integration into a microfluidic set-up for mixing and displacement of liquids, whereby the propulsion force and the resulting velocity regime can be modulated upon H2O2-controlled addition.

Radiochemistry on chip: towards dose-on-demand synthesis of PET radiopharmaceuticals.

We have developed an integrated microfluidic platform for producing 2-[(18)F]-fluoro-2-deoxy-D-glucose ((18)F-FDG) in continuous flow from a single bolus of radioactive isotope solution, with constant product yields achieved throughout the operation that were comparable to those reported for commercially available vessel-based synthesisers (40-80%). The system would allow researchers to obtain radiopharmaceuticals in a dose-on-demand setting within a few minutes. The flexible architecture of the platform, based on a modular design, can potentially be applied to the synthesis of other radiotracers that require a two-step synthetic approach, and may be adaptable to more complex synthetic routes by implementing additional modules. It can therefore be employed for standard synthesis protocols as well as for research and development of new radiopharmaceuticals.

Substituted pyrazolo[3,4-b]pyridines as human A1 adenosine antagonists: developments in understanding the receptor stereoselectivity.

A(1) adenosine receptor antagonists have been proposed to possess an interesting range of potential therapeutic applications. We have already reported the synthesis and the biological characterization of a family of pyrazolo[3,4-b]pyridine derivatives as A(1) adenosine ligands endowed with an antagonistic profile. In the present work, we report the LC separation of enantiomers of our most active A(1) antagonists together with the determination of their absolute configuration by means of X-ray crystal structure analysis. Biological assays confirmed a different activity for the two enantiomers, with the R one showing the higher human A(1)AR affinity. We also developed a homology model of this receptor subtype in order to suggest a binding disposition of the ligands into the hA(1)AR. All of the obtained data suggest that the compound's chirality plays a key role in A(1) affinity.

2-Hydroxypropyl-ß-cyclodextrin strongly improves water solubility and anti-proliferative activity of pyrazolo[3,4-d]pyrimidines Src-Abl dual inhibitors.

The main aim of this study was to enhance the solubility of pyrazolo[3,4-d]pyrimidines 1-8 able to strongly inhibit Src and Abl tyrosine kinase phosphorylation in cell-free assays and to significantly reduce leukemic and osteosarcoma cell lines growth, but characterized by very low solubility in aqueous media. Their water solubility was improved between 100 and 1000 folds by solubilization with 2-hydroxypropyl-ß-cyclodextrin (HPßCD) and ratio of inclusion complex were determined by phase solubility method. Finally, some complexed compounds were tested on different leukemic (K-652, KU-812 and HL-60) and osteosarcoma (SaOS-2) cell lines showing a good enhancement of biological response in comparison with the not complexed compounds.

Synthesis of new linear guanidines and macrocyclic amidinourea derivatives endowed with high antifungal activity against Candida spp. and Aspergillus spp.

New linear and cyclic guanidines were synthesized and tested in vitro for their antifungal activity toward clinically relevant strains of Candida species, in comparison to fluconazole. Macrocyclic compounds showed a minimum inhibitory concentration in the micromolar range and a biological activity profile in some cases better than that of fluconazole. One macrocyclic derivative was also tested against Aspergillus species and showed high antifungal activity comparable to that of amphotericin B and itraconazole.

Synthesis and biological evaluation of guanidino compounds endowed with subnanomolar affinity as competitive inhibitors of maize polyamine oxidase.

Previous studies on agmatine and its derivatives suggested that the presence of hydrophobic groups on the guanidine moiety was a crucial key for inhibitory activity of maize polyamine oxidase. Accordingly, new lipophilic agmatine and iminoctadine derivatives were synthesized and tested for their ability to inhibit this enzyme. Several compounds showed an affinity in the nanomolar range, while a cyclopropylmethyl derivative of iminoctadine was found to be the most potent inhibitor of maize polyamine oxidase reported so far (Ki = 0.08 nM).

Determination of permeability and lipophilicity of pyrazolo-pyrimidine tyrosine kinase inhibitors and correlation with biological data.

A library of 23 pyrazolo-pyrimidine compounds Src tyrosine kinase (TK) inhibitors, that reduced proliferation of a human osteogenic sarcoma cell line, was taken to investigate lack of correlation between inhibition of cellular viability (CV%) and enzymatic inhibition constants (K(i) Src). With the aim of understanding this behaviour, we focused on physico-chemical parameters which characterize partition coefficient and diffusion through membrane. Parallel artificial membrane permeability assay (PAMPA) has been frequently used for the evaluation of in vitro permeability of new chemical entities and, in this paper, a new approach for determining permeability of low soluble compounds was obtained. Goodness of PAMPA methodology was confirmed by logK(w) and computational approaches, by VolSurf, Cerius(2) and QikProp software programs. The results suggest that the lipophilicity and passive diffusion across the membranes do not significantly influence the activity of the compounds. This trend can be explained by a different target for some of the compounds in our set. In fact some compounds resulted also to be active toward Abl enzyme, another cytoplasmatic TK.

Substituted pyrazolo[3,4-b]pyridines as potent A1 adenosine antagonists: synthesis, biological evaluation, and development of an A1 bovine receptor model.

Sixty-eight new substituted pyrazolo[3,4-b]pyridine derivatives were synthesized and tested for enriching a library of active A(1) adenosine receptor (AR) antagonists belonging to the same class. These compounds were also used as an external test set to check the reliability of a 3D QSAR model recently reported by us. To investigate the binding mode of pyrazolopyridine derivatives, a model of the bovine A(1)AR (bA(1)AR) was developed by a novel homology modeling approach and used to evaluate the main interactions of the ligands with the receptor through docking studies. Results suggest important interactions of the ligands mainly with L3.33(88), T3.36(91), Q3.37(92) and H6.52(251), in agreement with mutagenesis data. The racemic mixture of the most active compound was separated into the corresponding enantiomers which showed a bA(1)AR affinity in the nanomolar range, with the R enantiomer sevenfold more active than the S enantiomer, according to results derived from calculations on the receptor model. Analysis of the bovine/human A(1)AR affinity profile of ligands supported the hypothesis that such receptors should be characterized by a different size of their binding site, responsible for the different affinity of the antagonists.