Nanofluidic-Based Accumulation of Antigens for Miniaturized Immunoassay.

The continuous advances of Nanofluidics have been stimulating the development of novel nanostructures and strategies to accumulate very diluted analytes, for implementing a new class of high sensitivity miniaturized polymeric sensors. We take advantage of the electrokinetic properties of these structures, which allow accumulating analytes inside asymmetric microfluidic structures to implement miniaturized sensors able to detect diluted solutions down to nearly 1.2 pg/mL. In particular, exploiting polydimethylsiloxane devices, fabricated by using the junction gap breakdown technique, we concentrate antigens inside a thin microfunnel functionalized with specific antibodies to favor the interaction and, if it is the case, the recognition between antigens in solution and antibodies anchored to the surface. The transduction mechanism consists in detecting the fluorescence signal of labeled avidin when it binds to biotinylated antigens. Here, we demonstrate that exploiting these electrokinetic phenomena, typical of nanofluidic structures, we succeeded in concentrating biomolecules in correspondence of a 1 pL sensing region, a strategy that grants to the device performance comparable to standard immunoassays.

Simultaneous Electro-Optical Tracking for Nanoparticle Recognition and Counting.

We present the first detailed experimental observation and analysis of nanoparticle electrophoresis through a nanochannel obtained with synchronous high-bandwidth electrical and camera recordings. Optically determined particle diffusion coefficients agree with values extracted from fitting electrical transport measurements to distributions from 1D Fokker-Planck diffusion-drift theory. This combined tracking strategy enables optical recognition and electrical characterization of nanoparticles in solution, which can have a broad range of applications in biology and materials science.

Improvement of Natural Polymeric Films Properties by Blend Formulation for Sustainable Active Food Packaging.

Active packaging manufactured with biopolymers extracted from agri-food waste is one of the most innovative and eco-sustainable strategies for maintaining food quality. However, biopolymers often present poor performances, which hinders their competitiveness compared with plastics. This work focused on developing and optimizing a natural polymeric blend produced by solvent casting based on zein and chitosan to improve the pure biopolymers' properties. The best results were obtained by blending zein and chitosan in a 1:2 weight ratio. The films were characterized in terms of morphology, mechanical and oxygen barrier properties, thermal stability, transparency and wettability. The blend production allowed us to obtain lower brittleness and lower stiffness materials compared with pure polymer films, with oxygen permeability values two orders of magnitude lower than pure zein, better optical properties with respect to pure chitosan and good thermal stability. The wettability properties of the blend did not result in being altered with respect to the single polymer, which was found to have hydrophilic behavior, highlighting the strong influence of glycerol used as a plasticizer. The results suggested that the polymer blending strategy is a viable and cost-effective method for producing packaging materials as alternatives to plastics.

Potential Use of PLA-Based Films Loaded with Antioxidant Agents from Spent Coffee Grounds for Preservation of Refrigerated Foods.

The aim of this work concerned the production of an active food packaging suitable for refrigerated foods. Polylactic-acid-based films were produced by optimizing the solvent casting technique and testing different loadings of extracts obtained from spent coffee grounds. Indeed, an extract obtained by high-pressure and -temperature extraction (HPTE) and a further purified extract by liquid-liquid extraction (LLE) were separately used as active agents, and the effects on packaging features and active compounds migration were analyzed. The selected active agents showed antioxidant and lipid peroxidation inhibition effects on food simulants (peroxide values of 9.2 ÷ 12.0 meqO2/kg extra virgin olive oil), demonstrating the possibility of enhancing food shelf life. In addition, significant effects on the packaging structure due to the presence of the extract were observed, since it can enhance gas barrier properties of the polymer (O2 permeability of 1.6 ÷ 1.3 × 10-9 cm2/s) and confer better processability. In general, the HPTE extract exhibited better performances than the further purified extract, which was due to the presence of a complex pool of antioxidants and the browning effect on the film but a limited loading capacity on the polymer (840 µg caffeine/g PLA), while higher loading capabilities were enabled using LLE extract.

Identification of the seeding mechanism in the spinodal instability of dewetting liquids.

HYPOTHESIS: Spinodal dewetting is one of the basic processes inducing a spontaneous withdrawal of a liquid from a substrate surface. In the accepted theory, thickness fluctuations generated by thermally activated capillary waves are amplified by the competing actions of surface tension and disjoining pressure. Ubiquitous sub-nanometric substrate roughness also produces thickness fluctuations and may play a role analogous but even more efficient in seeding the process. MODELLING: Analytic calculations valid at the early linear stage of the process and simulations extending the study to its whole non-linear development have been performed to compare features and the relative relevance of the two seeding mechanisms. FINDINGS: Calculations and simulations have shown that substrate roughness can replace capillary waves in seeding spinodal dewetting. A typically larger amplitude and a steady nature compared to the transitory one of capillary waves allow us to conclude that, contrary to the common view, substrate roughness is the prevailing seed of the spinodal instability. The consequence of our statement is that spinodal dewetting loses most of its stochastic nature and becomes, in principle, a process that can be tuned by engineering substrate roughness.

Size and functional tuning of solid state nanopores by chemical functionalization.

We demonstrate the possibility of using a simple functionalization procedure, based on an initial vapour-phase silanization, to control the size and functionality of solid state nanopores. The presented results show that, by varying the silanization time, it is possible to modify the efficiency of probe molecule attachment, thus shrinking the pore to the chosen size, while introducing a specific sensing selectivity. The proposed method allows us to tune the nanopore biosensor adapting it to the specific final application, and it can be efficiently applied when the pore initial diameter does not exceed a limit dimension related to the mean free path of the silane molecules at the working pressure.

Bevacizumab-Controlled Delivery from Polymeric Microparticle Systems as Interesting Tools for Pathologic Angiogenesis Diseases.

This work is a comparative study among three different biocompatible and biodegradable polymers, poly(lactic-co-glycolic acid), poly(ε-caprolactone), and poly(lactic acid), used to produce microparticles for the encapsulation of bevacizumab for drug delivery purposes. All the formulations were produced using the double emulsion water-oil-water evaporation method and characterized in terms of particle mean diameter, particle size distribution, and bevacizumab entrapment efficiency. Bevacizumab cumulative release was taken into consideration to study the dissolution kinetics from the three different polymeric delivery platforms for a period of 50 days at 37 °C in phosphate buffered saline and mathematical models of the drug release kinetic were attempted in order to describe the release phenomena from the different types of the studied microparticles. Finally, cell viability on human endothelial cell line EA.hy926 was studied to define the maximum cytocompatible concentration for each microsystem, registering the mitochondrial functionality through MTS assay.

A Two-Step Approach to Tune the Micro and Nanoscale Morphology of Porous Niobium Oxide to Promote Osteointegration.

We present a two-step surface modification process to tailor the micro and nano morphology of niobium oxide layers. Niobium was firstly anodized in spark regime in a Ca- and P-containing solution and subsequently treated by acid etching. The effects of anodizing time and applied potential on the surface morphology is investigated with SEM and AFM, complemented by XPS compositional analysis. Anodizing with a limiting potential of 250 V results in the fast growth of oxide layers with a homogeneous distribution of micro-sized pores. Cracks are, however, observed on 250 V grown layers. Limiting the anodizing potential to 200 V slows down the oxide growth, increasing the anodizing time needed to achieve a uniform pore coverage but produces fracture-free oxide layers. The surface nano morphology is further tuned by a subsequent acid etching process that leads to the formation of nano-sized pits on the anodically grown oxide surface. In vitro tests show that the etching-induced nanostructure effectively promotes cell adhesion and spreading onto the niobium oxide surface.

High blood flow shear stress values are associated with circulating tumor cells cluster disaggregation in a multi-channel microfluidic device.

Metastasis represents a dynamic succession of events involving tumor cells which disseminate through the organism via the bloodstream. Circulating tumor cells (CTCs) can flow the bloodstream as single cells or as multicellular aggregates (clusters), which present a different potential to metastasize. The effects of the bloodstream-related physical constraints, such as hemodynamic wall shear stress (WSS), on CTC clusters are still unclear. Therefore, we developed, upon theoretical and CFD modeling, a new multichannel microfluidic device able to simultaneously reproduce different WSS characterizing the human circulatory system, where to analyze the correlation between SS and CTC clusters behavior. Three physiological WSS levels (i.e. 2, 5, 20 dyn/cm2) were generated, reproducing values typical of capillaries, veins and arteries. As first validation, triple-negative breast cancer cells (MDA-MB-231) were injected as single CTCs showing that higher values of WSS are correlated with a decreased viability. Next, the SS-mediated disaggregation of CTC clusters was computationally investigated in a vessels-mimicking domain. Finally, CTC clusters were injected within the three different circuits and subjected to the three different WSS, revealing that increasing WSS levels are associated with a raising clusters disaggregation after 6 hours of circulation. These results suggest that our device may represent a valid in vitro tool to carry out systematic studies on the biological significance of blood flow mechanical forces and eventually to promote new strategies for anticancer therapy.

In vitro demonstration of intestinal absorption mechanisms of different sugars using 3D organotypic tissues in a fluidic device.

Intestinal permeability is crucial in regulating the bioavailability and, consequently, the biological effects of drugs and compounds. However, systematic and quantitative studies of the absorption of molecules are quite limited due to a lack of reliable experimental models able to mimic human in vivo responses. In this work, we present an in vitro perfused model of the small intestinal barrier using a 3D reconstructed intestinal epithelium integrated into a fluid-dynamic biore­actor (MIVO®) resembling the physiological stimuli of the intestinal environment. This platform was investigated in both healthy and induced pathological conditions by monitoring the absorption of two non-metabolized sugars, lactulose and mannitol, frequently used as indicators of intestinal barrier dysfunctions. In healthy conditions, an in vivo-like plateau of the percentage of absorbed sugars was reached, where mannitol absorption was much greater than lactulose absorption. Moreover, a model of pathologically altered intestinal permeability was generated by depleting extracellular Ca2+, using a calcium-specific chelator. After calcium depletion, the pattern of sugar passage observed under pathological conditions was reversed only in dynamic conditions in the MIVO® chamber, due to the dynamic fluid flow beneath the membrane, but not in static conditions. Therefore, the combination of the MIVO® with the EpiIntestinal™ platform can rep­resent a reliable in vitro model to study the passage of molecules across the healthy or pathological small intestinal barrier by discriminating the two main mechanisms of intestinal absorption.

Integrating Microstructured Electrospun Scaffolds in an Open Microfluidic System for in Vitro Studies of Human Patient-Derived Primary Cells.

Recent studies have suggested that microenvironmental stimuli play a significant role in regulating cellular proliferation and migration, as well as in modulating self-renewal and differentiation processes of mammary cells with stem cell (SCs) properties. Recent advances in micro/nanotechnology and biomaterial synthesis/engineering currently enable the fabrication of innovative tissue culture platforms suitable for maintenance and differentiation of SCs in vitro. Here, we report the design and fabrication of an open microfluidic device (OMD) integrating removable poly(ε-caprolactone) (PCL) based electrospun scaffolds, and we demonstrate that the OMD allows investigation of the behavior of human cells during in vitro culture in real time. Electrospun scaffolds with modified surface topography and chemistry can influence attachment, proliferation, and differentiation of mammary SCs and epigenetic mechanisms that maintain luminal cell identity as a function of specific morphological or biochemical cues imparted by tailor-made fiber post-treatments. Meanwhile, the OMD architecture allows control of cell seeding and culture conditions to collect more accurate and informative in vitro assays. In perspective, integrated systems could be tailor-made to mimic specific physiological conditions of the local microenvironment and then analyze the response from screening specific drugs for more effective diagnostics, long-term prognostics, and disease intervention in personalized medicine.

Increased Flexibility in Lab-on-Chip Design with a Polymer Patchwork Approach.

Nanofluidic structures are often the key element of many lab-on-chips for biomedical and environmental applications. The demand for these devices to be able to perform increasingly complex tasks triggers a request for increasing the performance of the fabrication methods. Soft lithography and poly(dimethylsiloxane) (PDMS) have since long been the basic ingredients for producing low-cost, biocompatible and flexible devices, replicating nanostructured masters. However, when the desired functionalities require the fabrication of shallow channels, the "roof collapse" phenomenon, that can occur when sealing the replica, can impair the device functionalities. In this study, we demonstrate that a "focused drop-casting" of h-PDMS (hard PDMS) on nanostructured regions, provides the necessary stiffness to avoid roof collapse, without increasing the probability of deep cracks formation, a drawback that shows up in the peel-off step, when h-PDMS is used all over the device area. With this new approach, we efficiently fabricate working devices with reproducible sub-100 nm structures. We verify the absence of roof collapse and deep cracks by optical microscopy and, in order to assess the advantages that are introduced by the proposed technique, the acquired images are compared with those of cracked devices, whose top layer, of h-PDMS, and with those of collapsed devices, made of standard PDMS. The geometry of the critical regions is studied by atomic force microscopy of their resin casts. The electrical resistance of the nanochannels is measured and shown to be compatible with the estimates that can be obtained from the geometry. The simplicity of the method and its reliability make it suitable for increasing the fabrication yield and reducing the costs of nanofluidic polymeric lab-on-chips.

The Role of Surfaces in Gas Transport Through Polymer Membranes.

This paper describes a procedure to measure the permeability P, diffusivity D, and rate of adsorption k1, thus determining the solubility S and rate of desorption k2 of He, N2, O2, CH4, and CO2 on a polydimethylsiloxane (PDMS) membrane. The described procedure is able to determine experimentally all the physical quantities that characterize the gas transport process through a thin rubber polymer membrane. The experiments were carried out at room temperature and at a transmembrane pressure of 1 atm. The results are in good agreement with the available data in the literature and offer an evaluation of k1 and k2.

Nanotechnology applications in medicine.

In recent years there has been a rapid increase in nanotechnology applications to medicine in order to prevent and treat diseases in the human body. The established and future applications have the potential to dramatically change medical science. The present paper will give a few examples that could transform common medical procedures.

Gas permeation through rubbery polymer nano-corrugated membranes.

The purpose of this investigation is to fabricate PDMS membranes with reliable surface roughness in order to reduce the surface resistances and to study its impact on the permeation rate. The permeance of CO2 through PDMS membranes with rough surfaces at nanoscale is studied and compared with the one of membranes with flat surfaces. At very low thickness, rough membranes have a permeance greater than that of membranes with flat surfaces. The enhancement occurs in a regime where the gas transport is sorption desorption surface rate limited, and cannot be explained by the increase in surface area due to the corrugation. The analysis, introducing a phenomenological model in analogy with electrical flow, indicates that nano-corrugation reduces the surface resistance. To test the model, the permeance of N2 is also measured in the same experimental conditions and the influence of surface roughness on permeation rate of CO2, He, CH4 and N2 is studied. The comparison among the gases suggests that the Henry's coefficient depends on the surface roughness and allows discussing the role of roughness on membrane selectivity.

Selective protein detection with a dsLNA-functionalized nanopore.

In the last years, nanopore technology has been increasingly exploited for biomolecule detection and analysis. Recently, the main focus of the research has moved from the study of nucleic acids to the analysis of proteins and DNA-protein complexes. In this paper, chemically functionalized solid-state nanopore has been used to recognize Nuclear Factor-kappa B proteins (NF-κB), that are involved in several disorders and inflammation processes, so that their identification is of crucial importance for prognostic applications. In particular, we show that it is possible to electrically detect the specific interaction between p50, a protein belonging to the NF-κB family, and dsLNA probe molecules covalently attached to the surface of a FIB fabricated SiN pore. The obtained results have been compared with those related to BSA protein, which does not interact with the used probes. Finally, the potential of the device has been further tested by analyzing a whole cell extract. In this case, three principal peaks in the distribution of electrical event duration can be identified, corresponding to different interacting NF-κB complexes, so that the methodology appears to be effective also to study biological samples of considerable complexity. Ultimately, the presented data emphasize the selectivity and versatility of the functionalized nanopore device, demonstrating its applicability in bioanalytics and advanced diagnostics.

Stretching of DNA confined in nanochannels with charged walls.

There is currently a growing interest in control of stretching of DNA inside nanoconfined regions due to the possibility to analyze and manipulate single biomolecules for applications such as DNA mapping and barcoding, which are based on stretching the DNA in a linear fashion. In the present work, we couple Finite Element Methods and Monte Carlo simulations in order to study the conformation of DNA molecules confined in nanofluidic channels with neutral and charged walls. We find that the electrostatic forces become more and more important when lowering the ionic strength of the solution. The influence of the nanochannel cross section geometry is also studied by evaluating the DNA elongation in square, rectangular, and triangular channels. We demonstrate that coupling electrostatically interacting walls with a triangular geometry is an efficient way to stretch DNA molecules at the scale of hundreds of nanometers. The paper reports experimental observations of λ-DNA molecules in poly(dimethylsiloxane) nanochannels filled with solutions of different ionic strength. The results are in good agreement with the theoretical predictions, confirming the crucial role of the electrostatic repulsion of the constraining walls on the molecule stretching.

Order versus Disorder: in vivo bone formation within osteoconductive scaffolds.

In modern biomaterial design the generation of an environment mimicking some of the extracellular matrix features is envisaged to support molecular cross-talk between cells and scaffolds during tissue formation/remodeling. In bone substitutes chemical biomimesis has been particularly exploited; conversely, the relevance of pre-determined scaffold architecture for regenerated bone outputs is still unclear. Thus we aimed to demonstrate that a different organization of collagen fibers within newly formed bone under unloading conditions can be generated by differently architectured scaffolds. An ordered and confined geometry of hydroxyapatite foams concentrated collagen fibers within the pores, and triggered their self-assembly in a cholesteric-banded pattern, resulting in compact lamellar bone. Conversely, when progenitor cells were loaded onto nanofibrous collagen-based sponges, new collagen fibers were distributed in a nematic phase, resulting mostly in woven isotropic bone. Thus specific biomaterial design relevantly contributes to properly drive collagen fibers assembly to target bone regeneration.

Modulating DNA translocation by a controlled deformation of a PDMS nanochannel device.

Several strategies have been developed for the control of DNA translocation in nanopores and nanochannels. However, the possibility to reduce the molecule speed is still challenging for applications in the field of single molecule analysis, such as ultra-rapid sequencing. This paper demonstrates the possibility to alter the DNA translocation process through an elastomeric nanochannel device by dynamically changing its cross section. More in detail, nanochannel deformation is induced by a macroscopic mechanical compression of the polymeric device. This nanochannel squeezing allows slowing down the DNA molecule passage inside it. This simple and low cost method is based on the exploitation of the elastomeric nature of the device, can be coupled with different sensing techniques, is applicable in many research fields, such as DNA detection and manipulation, and is promising for further development in sequencing technology.

DNA manipulation with elastomeric nanostructures fabricated by soft-moulding of a FIB-patterned stamp.

A Focused Ion Beam (FIB)-patterned silicon mould is used to fabricate elastomeric nanostructures, whose cross-section can be dynamically and reversibly tuned by applying a controlled mechanical stress. Direct-write, based on FIB milling, allows the fabrication of nanostructures with a variety of different geometries, aspect ratio, spacing and distribution offering a higher flexibility compared to other nanopatterning approaches. Moreover, a simple double replication process based on poly(dimethylsiloxane) permits a strong reduction of the fabrication costs that makes this approach well-suited for the production of low cost nanofluidic devices. DNA stretching and single molecule manipulation capabilities of these platforms have been successfully demonstrated.