Cyclic olefin copolymer (COC) as a promising biomaterial for affecting bacterial colonization: investigation on Vibrio campbellii.

Cyclic olefin copolymer (COC) has emerged as an interesting biocompatible material for Organ-on-a-Chip (OoC) devices monitoring growth, viability, and metabolism of cells. Despite ISO 10993 approval, systematic investigation of bacteria grown onto COC is a still not documented issue. This study discusses biofilm formations of the canonical wild type BB120 Vibrio campbellii strain on a native COC substrate and addresses the impact of the physico-chemical properties of COC compared to conventional hydroxyapatite (HA) and poly(dimethylsiloxane) (PDMS) surfaces. An interdisciplinary approach combining bacterial colony counting, light microscopy imaging and advanced digital image processing remarks interesting results. First, COC can reduce biomass adhesion with respect to common biopolymers, that is suitable for tuning biofilm formations in the biological and medical areas. Second, remarkably different biofilm morphology (dendritic complex patterns only in the case of COC) was observed among the examined substrates. Third, the observed biofilm morphogenesis was related to the interaction of COC with the conditioning layer of the planktonic biological medium. Fourth, Level Co-occurrence Matrix (CGLM)-based analysis enabled quantitative assessment of the biomass textural fractal development under different coverage conditions. All of this is of key practical relevance in searching innovative biocompatible materials for pharmaceutical, implantable and medical products.

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.

Glass Microdroplet Generator for Lipid-Based Double Emulsion Production.

Microfluidics offers a highly controlled and reproducible route to synthesize lipid vesicles. In recent years, several microfluidic approaches have been introduced for this purpose, but double emulsions, such as Water-in-Oil-in-Water (W/O/W) droplets, are preferable to produce giant vesicles that are able to maximize material encapsulation. Flow focusing (FF) is a technique used to generate double emulsion droplets with high monodispersity, a controllable size, and good robustness. Many researchers use polydimethylsiloxane as a substrate material to fabricate microdroplet generators, but it has some limitations due to its hydrophobicity, incompatibility with organic solvents, and the molecular adsorption on the microchannel walls. Thus, specific surface modification and functionalization steps, which are uncomfortable to perform in closed microchannels, are required to overcome these shortcomings. Here, we propose glass as a material to produce a chip with a six-inlet junction geometry. The peculiar geometry and the glass physicochemical properties allow for W/O/W droplet formation without introducing microchannel wall functionalization and using a variety of reagents and organic solvents. The robust glass chip can be easily cleaned and used repeatedly, bringing advantages in terms of cost and reproducibility in emulsion preparation.

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.

MEED: A novel robust contrast enhancement procedure yielding highly-convergent thresholding of biofilm images.

Morphological and statistical investigation of biofilm images may be even more critical than the image acquisition itself, in particular in the presence of morphologically complex distributions, due to the unavoidable impact of the measurement technique too. Hence, digital image pre-processing is mandatory for reliable feature extraction and enhancement preliminary to segmentation. Also, pattern recognition in automated deep learning (both supervised and unsupervised) models often requires a preliminary effective contrast-enhancement. However, no universal consensus exists on the optimal contrast enhancement approach. This paper presents and discusses a new general, robust, reproducible, accurate and easy to implement contrast enhancement procedure, briefly named MEED-procedure, able to work on images with different bacterial coverages and biofilm structures, coming from different imaging instrumentations (herein stereomicroscope and transmission microscope). It exploits a proper succession of basic morphological operations (erosion and dilation) and a horizontal line structuring element, to minimize the impact on size and shape of the even finer bacterial features. It systematically enhances the objects of interest, without histogram stretching and/or undesirable artifacts yielded by common automated methods. The quality of the MEED-procedure is ascertained by segmentation tests which demonstrate its robustness regarding the determination of threshold and convergence of the thresholding algorithm. Extensive validation tests over a rich image database, comparison with the literature and comprehensive discussion of the conceptual background support the superiority of the MEED-procedure over the existing methods and demonstrate it is not a routine application of morphological operators.

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.

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.

Lab-on-Chip for Exosomes and Microvesicles Detection and Characterization.

Interest in extracellular vesicles and in particular microvesicles and exosomes, which are constitutively produced by cells, is on the rise for their huge potential as biomarkers in a high number of disorders and pathologies as they are considered as carriers of information among cells, as well as being responsible for the spreading of diseases. Current methods of analysis of microvesicles and exosomes do not fulfill the requirements for their in-depth investigation and the complete exploitation of their diagnostic and prognostic value. Lab-on-chip methods have the potential and capabilities to bridge this gap and the technology is mature enough to provide all the necessary steps for a completely automated analysis of extracellular vesicles in body fluids. In this paper we provide an overview of the biological role of extracellular vesicles, standard biochemical methods of analysis and their limits, and a survey of lab-on-chip methods that are able to meet the needs of a deeper exploitation of these biological entities to drive their use in common clinical practice.

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.

Quartz Crystal Microbalance as Cell-Based Biosensor to Detect and Study Cytoskeletal Alterations and Dynamics.

Several techniques can be used to monitor cell dynamism after a perturbation. Among these, Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D) offers the great advantage to study the mechanical properties of cells in real-time and with a great sensitivity. Here, we used QCM-D to investigate the effects of two cytoskeleton-targeting agents, cytochalasin D (CytoD) and Y27632, on human MCF-7 cells. Cell adhesion on the sensor surface, crucial for in-flow experiments, was obtained by covalent adsorption of a fibronectin (FN) film, an extracellular matrix (ECM) protein. Direct analysis of MCF-7 cells on FN-coated sensor, shows a specific cellular response that was revealed and quantified by QCM-D after drugs exposure. Notably, upon treatment with Y27632, we observed a two-regime dissipation behavior that we associated with specific modifications of actin filaments and signaling proteins providing a link between biophysical and molecular mechanisms. Overall, this approach opens new opportunities for studying cellular response to mechanical cues in different biological conditions.

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.

Quartz crystal microbalance with dissipation (QCM-D) as tool to exploit antigen-antibody interactions in pancreatic ductal adenocarcinomadetection.

Novel synthetic peptides represent smart molecules for antigen-antibody interactions in several bioanalytics applications, from purification to serum screening. Their immobilization onto a solid phase is considered a key point for sensitivity increasing. In this view, we exploited Quartz Crystal Microbalance with simultaneous frequency and dissipation monitoring (QCM-D) with a double aim, specifically, as investigative tool for spacers monolayer assembling and its functional evaluation, as well as high sensitive method for specific immunosorbent assays. The method was applied to pancreatic ductal adenocarcinoma (PDAC) detection by studying the interactions between synthetic phosphorylated and un-phosphorylated α-enolase peptides with sera of healthy and PDAC patients. The synthetic peptides were immobilized on the gold surface of the QCM-D sensor via a self-assembled alkanethiol monolayer. The presented experimental results can be applied to the development of surfaces less sensitive to non-specific interactions with the final target to suggest specific protocols for detecting PDAC markers with un-labeled biosensors.

Toward Quantum-dot Cellular Automata units: thiolated-carbazole linked bisferrocenes.

Quantum-dot Cellular Automata (QCA) exploit quantum confinement, tunneling and electrostatic interaction for transistorless digital computing. Implementation at the molecular scale requires carefully tailored units which must obey several structural and functional constraints, ranging from the capability to confine charge efficiently on different 'quantum-dot centers'-in order to sharply encode the Boolean states-up to the possibility of having their state blanked out upon application of an external signal. In addition, the molecular units must preserve their geometry in the solid state, to interact electrostatically in a controlled way. Here, we present a novel class of organometallic molecules, 6-3,6-bis(1-ethylferrocen)-9H-carbazol-9-yl-6-hexan-1-thiols, which are engineered to satisfy all such crucial requirements at once, as confirmed by electrochemistry and scanning tunneling microscopy measurements, and first principles density functional calculations.