A miniaturized multicellular platform to mimic the 3D structure of the alveolar-capillary barrier.

Several diseases affect the alveoli, and the efficacy of medical treatments and pharmaceutical therapies is hampered by the lack of pre-clinical models able to recreate in vitro the diseases. Microfluidic devices, mimicking the key structural and compositional features of the alveoli, offer several advantages to medium and high-throughput analysis of new candidate therapies. Here, we developed an alveolus-on-a-chip recapitulating the microanatomy of the physiological tissue by including the epithelium, the fibrous interstitial layer and the capillary endothelium. A PDMS device was obtained assembling a top layer and a bottom layer obtained by replica molding. A polycaprolactone/gelatin (PCL-Gel) electrospun membrane was included within the two layers supporting the seeding of 3 cell phenotypes. Epithelial cells were grown on a fibroblast-laden collagen hydrogel located on the top side of the PCL-Gel mats while endothelial cells were seeded on the basolateral side of the membrane. The innovative design of the microfluidic device allows to replicate both cell-cell and cell-extracellular matrix interactions according to the in vivo cell arrangement along with the establishment of physiologically relevant air-liquid interface conditions. Indeed, high cell viability was confirmed for up to 10 days and the formation of a tight endothelial and epithelial barrier was assessed by immunofluorescence assays.

Organic Electrochemical Transistor Immuno-Sensors for Spike Protein Early Detection.

The global COVID-19 pandemic has had severe consequences from the social and economic perspectives, compelling the scientific community to focus on the development of effective diagnostics that can combine a fast response and accurate sensitivity/specificity performance. Presently available commercial antigen-detecting rapid diagnostic tests (Ag-RDTs) are very fast, but still face significant criticisms, mainly related to their inability to amplify the protein signal. This translates to a limited sensitive outcome and, hence, a reduced ability to hamper the spread of SARS-CoV-2 infection. To answer the urgent need for novel platforms for the early, specific and highly sensitive detection of the virus, this paper deals with the use of organic electrochemical transistors (OECTs) as very efficient ion-electron converters and amplifiers for the detection of spike proteins and their femtomolar concentration. The electrical response of the investigated OECTs was carefully analyzed, and the changes in the parameters associated with the transconductance (i.e., the slope of the transfer curves) in the gate voltage range between 0 and 0.3 V were found to be more clearly correlated with the spike protein concentration. Moreover, the functionalization of OECT-based biosensors with anti-spike and anti-nucleocapside proteins, the major proteins involved in the disease, demonstrated the specificity of these devices, whose potentialities should also be considered in light of the recent upsurge of the so-called "long COVID" syndrome.

Immuno-Sensing at Ultra-Low Concentration of TG2 Protein by Organic Electrochemical Transistors.

Transglutaminase 2 (TG2) is a ubiquitously expressed member of the transglutaminase family with Ca2+-dependent protein crosslinking activity. Its subcellular localization is crucial in determining its function, and indeed, TG2 is found in the extracellular matrix, mitochondria, recycling endosomes, plasma membrane, cytosol, and nucleus because it is associated with cell growth, differentiation, and apoptosis. It is involved in several pathologies, such as celiac disease, cardiovascular, hepatic, renal, and fibrosis diseases, carrying out opposite functions of up and down regulation in the progression of the same pathology. Therefore, this fine regulation requires a very sensitive and specific method of identification of TG2, which is to be detected in very small quantities in a deregulated condition. Here, we demonstrate the possibility of detecting TG2 down to attomolar concentration by using organic electrochemical transistors driven by gold electrodes functionalized with anti-TG2 antibodies. In particular, a direct correlation between the TG2 concentration and the transistor transconductance values, as extracted from typical transfer curves, was found. Overall, our findings highlight the potentialities of this new biosensing approach for the detection of TG2 in the context of pathological diseases, offering a rapid and cost-effective alternative to traditional methods.

Pumpless deterministic lateral displacement separation using a paper capillary wick.

Deterministic lateral displacement (DLD) is a passive separation method that separates particles by hydrodynamic size. This label-free method is a promising technique for cell separation because of its high size resolution and insensitivity to flow rate. Development of capillary-driven microfluidic technologies allows microfluidic devices to be operated without any external power for fluid pumping, lowering their total cost and complexity. Herein, we develop and test a DLD-based particle and cell sorting method that is driven entirely by capillary pressure. We show microchip self-filling, flow focusing, flow stability, and capture of separated particles. We achieve separation efficiency of 92% for particle-particle separation and more than 99% efficiency for cell-particle separation. The high performance of driven flow and separation along with simplicity of the operation and setup make it a valuable candidate for point-of-care devices.

Organic Bioelectronics Development in Italy: A Review.

In recent years, studies concerning Organic Bioelectronics have had a constant growth due to the interest in disciplines such as medicine, biology and food safety in connecting the digital world with the biological one. Specific interests can be found in organic neuromorphic devices and organic transistor sensors, which are rapidly growing due to their low cost, high sensitivity and biocompatibility. This trend is evident in the literature produced in Italy, which is full of breakthrough papers concerning organic transistors-based sensors and organic neuromorphic devices. Therefore, this review focuses on analyzing the Italian production in this field, its trend and possible future evolutions.

PDAC-on-chip for in vitro modeling of stromal and pancreatic cancer cell crosstalk.

Pancreatic ductal adenocarcinoma (PDAC) mainly develops in the head of the pancreas, within the acino-ductal unit composed of acinar and ductal cells surrounded by pancreatic stellate cells (PSCs). PSCs strongly influence the tumor microenvironment by triggering an intense stromal deposition, which plays a key role in tumor progression and limits drug perfusion. We have developed a microfluidic in vitro model recreating the in vivo tumor-stroma crosstalk to replicate the steps of PDAC evolution towards the establishment of an efficient in vitro platform for innovative therapy validation. The multilayer PDAC-on-chip was designed to culture the PDAC cells and the PSCs embedded in a type I collagen gel in the top and bottom layers, respectively. The presence of a biomimetic nanofibrous membrane in the middle of the chip permits the control of interactions between the two cell lines and the easy analysis of the effects of the crosstalk on cell behavior. First, the PDAC-stromal cell relationship was evaluated under co-culture conditions on 24-well inserts including the PCL/Gel electrospun membrane. This simplified model shows that human fibroblasts change their morphology and secrete larger amounts of IL-6 cytokines in the presence of tumor cells, confirming the activation of stromal cells under co-culture. Then, the PDAC-on-chip system was validated by demonstrating that human fibroblasts seeded in a 3D collagen matrix in the bottom microchannel also change to a myofibroblast-like shape with increased expression of α-SMA and secrete larger amounts of IL-6 cytokines. This microfluidic system is suitable for the evaluation of drug efficacy and serves as a powerful tool for understanding the early evolution steps of PDAC.

Design of a Portable Microfluidic Platform for EGOT-Based in Liquid Biosensing.

In biosensing applications, the exploitation of organic transistors gated via a liquid electrolyte has increased in the last years thanks to their enormous advantages in terms of sensitivity, low cost and power consumption. However, a practical aspect limiting the use of these devices in real applications is the contamination of the organic material, which represents an obstacle for the realization of a portable sensing platform based on electrolyte-gated organic transistors (EGOTs). In this work, a novel contamination-free microfluidic platform allowing differential measurements is presented and validated through finite element modeling simulations. The proposed design allows the exposure of the sensing electrode without contaminating the EGOT device during the whole sensing tests protocol. Furthermore, the platform is exploited to perform the detection of bovine serum albumin (BSA) as a validation test for the introduced differential protocol, demonstrating the capability to detect BSA at 1 pM concentration. The lack of contamination and the differential measurements provided in this work can be the first steps towards the realization of a reliable EGOT-based portable sensing instrument.

Organic electrochemical transistors as novel biosensing platforms to study the electrical response of whole blood and plasma.

In this paper, for the first time to the best of our knowledge, organic electrochemical transistors are employed to investigate the electrical response of human blood, plasma and alternative buffer solutions that inhibit red blood cell (RBC) aggregation. Our focus is on selecting a suitable electrolytic platform and the related operating conditions, where the RBC effect on the OECT response can be observed separately from the strong ionic environment of plasma in whole blood. The transient response of whole blood to pulse experiments is characterized by two time constants, which can be related to blood viscosity and to the capacitive coupling between the ionic and electronic components of the overall system. The role of capacitive effects, likely due to enhanced double-layer formation by negatively charged RBCs, is also confirmed by the increase of transconductance which was found in RBC suspensions as compared to the suspending buffer. Overall, the complex behavior found in these experiments provides new insights for the development of innovative blood-based sensing devices for biomedical applications.

Focalization Performance Study of a Novel Bulk Acoustic Wave Device.

This work illustrates focalization performances of a silicon-based bulk acoustic wave device applied for the separation of specimens owing to micrometric dimensions. Samples are separated in the microfluidic channel by the presence of an acoustic field, which focalizes particles or cells according to their mechanical properties compared to the surrounded medium ones. Design and fabrication processes are reported, followed by focalization performance tests conducted either with synthetic particles or cells. High focalization performances occurred at different microparticle concentrations. In addition, preliminary tests carried out with HL-60 cells highlighted an optimal separation performance at a high flow rate and when cells are mixed with micro and nanoparticles without affecting device focalization capabilities. These encouraging results showed how this bulk acoustic wave device could be exploited to develop a diagnostic tool for early diagnosis or some specific target therapies by separating different kinds of cells or biomarkers possessing different mechanical properties such as shapes, sizes and densities.

Layered Double Hydroxide-Based Gas Sensors for VOC Detection at Room Temperature.

Miniaturized low-cost sensors for volatile organic compounds (VOCs) have the potentiality to become a fundamental tool for indoor and outdoor air quality monitoring, to significantly improve everyday life. Layered double hydroxides (LDHs) belong to the class of anionic clays and are largely employed for NO x detection, while few results are reported on VOCs. In this work, a novel LDH coprecipitation method is proposed. For the first time, a study comparing four LDHs (ZnAl-Cl, ZnFe-Cl, ZnAl-NO3, and MgAl-NO3) is carried out to investigate the sensing performances. As explored through several microscopy and spectroscopy analyses, LDHs show a morphology characterized by a large surface area and a three-dimensional hierarchical flowerlike architecture with micro- and nanopores that induce a fast diffusion and highly effective surface interaction of the target gases. The fabricated sensors, operating at room temperature, are able to reversibly and selectively detect acetone, ethanol, ammonia, and chlorine vapors, reaching significant sensing response values up to 6% at 21 °C. The results demonstrate that by changing the LDHs' composition, it is possible to modulate the sensitivity and selectivity of the sensor, helping the discrimination of different analytes, and the consequent integration on a sensor array paves the way for electronic nose development.

Interfacing aptamers, nanoparticles and graphene in a hierarchical structure for highly selective detection of biomolecules in OECT devices.

In several biomedical applications, the detection of biomarkers demands high sensitivity, selectivity and easy-to-use devices. Organic electrochemical transistors (OECTs) represent a promising class of devices combining a minimal invasiveness and good signal transduction. However, OECTs lack of intrinsic selectivity that should be implemented by specific approaches to make them well suitable for biomedical applications. Here, we report on a biosensor in which selectivity and a high sensitivity are achieved by interfacing, in an OECT architecture, a novel gate electrode based on aptamers, Au nanoparticles and graphene hierarchically organized to optimize the final response. The fabricated biosensor performs state of the art limit of detection monitoring biomolecules, such as thrombin-with a limit of detection in the picomolar range (≤ 5 pM) and a very good selectivity even in presence of supraphysiological concentrations of Bovine Serum Albumin (BSA-1mM). These accomplishments are the final result of the gate hierarchic structure that reduces sterich indrance that could contrast the recognition events and minimizes false positive, because of the low affinity of graphene towards the physiological environment. Since our approach can be easily applied to a large variety of different biomarkers, we envisage a relevant potential for a large series of different biomedical applications.

Editorial for the Special Issue on 2D Nanomaterials Processing and Integration in Miniaturized Devices.

Initially considered little more than a scientific curiosity, the family of 2D nanomaterials has become increasingly popular over the last decade [...].

Effect of Volatile Organic Compounds Adsorption on 3D-Printed PEGDA:PEDOT for Long-Term Monitoring Devices.

We report on the preparation and stereolithographic 3D printing of a resin based on the composite between a poly(ethylene glycol) diacrylate (PEGDA) host matrix and a poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) (PEDOT:PSS) filler, and the related cumulative volatile organic compounds' (VOCs) adsorbent properties. The control of all the steps for resin preparation and printing through morphological (SEM), structural (Raman spectroscopy) and functional (I/V measurements) characterizations allowed us to obtain conductive 3D objects of complex and reproducible geometry. These systems can interact with chemical vapors in the long term by providing a consistent and detectable variation of their structural and conductive characteristics. The materials and the manufacture protocol here reported thus propose an innovative and versatile technology for VOCs monitoring systems based on cumulative adsorption effects.

Rapid prototyping of 3D Organic Electrochemical Transistors by composite photocurable resin.

Rapid Prototyping (RP) promises to induce a revolutionary impact on how the objects can be produced and used in industrial manufacturing as well as in everyday life. Over the time a standard technique as the 3D Stereolithography (SL) has become a fundamental technology for RP and Additive Manufacturing (AM), since it enables the fabrication of the 3D objects from a cost-effective photocurable resin. Efforts to obtain devices more complex than just a mere aesthetic simulacre, have been spent with uncertain results. The multidisciplinary nature of such manufacturing technique furtherly hinders the route to the fabrication of complex devices. A good knowledge of the bases of material science and engineering is required to deal with SL technological, characterization and testing aspects. In this framework, our study aims to reveal a new approach to obtain RP of complex devices, namely Organic Electro-Chemical Transistors (OECTs), by SL technique exploiting a resin composite based on the conductive poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) and the photo curable Poly(ethylene glycol) diacrylate (PEGDA). A comprehensive study is presented, starting from the optimization of composite resin and characterization of its electrochemical properties, up to the 3D OECTs printing and testing. Relevant performances in biosensing for dopamine (DA) detection using the 3D OECTs are reported and discussed too.

Application of a Micro Free-Flow Electrophoresis 3D Printed Lab-on-a-Chip for Micro-Nanoparticles Analysis.

The present work describes a novel microfluidic free-flow electrophoresis device developed by applying three-dimensional (3D) printing technology to rapid prototype a low-cost chip for micro- and nanoparticle collection and analysis. Accurate reproducibility of the device design and the integration of the inlet and outlet ports with the proper tube interconnection was achieved by the additive manufacturing process. Test prints were performed to compare the glossy and the matte type of surface finish. Analyzing the surface topography of the 3D printed device, we demonstrated how the best reproducibility was obtained with the glossy device showing a 5% accuracy. The performance of the device was demonstrated by a free-flow zone electrophoresis application on micro- and nanoparticles with different dimensions, charge surfaces and fluorescent dyes by applying different separation voltages up to 55 V. Dynamic light scattering (DLS) measurements and ultraviolet-visible spectroscopy (UV-Vis) analysis were performed on particles collected at the outlets. The percentage of particles observed at each outlet was determined in order to demonstrate the capability of the micro free-flow electrophoresis (µFFE) device to work properly in dependence of the applied electric field. In conclusion, we rapid prototyped a microfluidic device by 3D printing, which ensured micro- and nanoparticle deviation and concentration in a reduced operation volume and hence suitable for biomedical as well as pharmaceutical applications.

Electrospun Nanofibers: from Food to Energy by Engineered Electrodes in Microbial Fuel Cells.

Microbial fuel cells (MFCs) are bio-electrochemical devices able to directly transduce chemical energy, entrapped in an organic mass named fuel, into electrical energy through the metabolic activity of specific bacteria. During the last years, the employment of bio-electrochemical devices to study the wastewater derived from the food industry has attracted great interest from the scientific community. In the present work, we demonstrate the capability of exoelectrogenic bacteria used in MFCs to catalyze the oxidation reaction of honey, employed as a fuel. With the main aim to increase the proliferation of microorganisms onto the anode, engineered electrodes are proposed. Polymeric nanofibers, based on polyethylene oxide (PEO-NFs), were directly electrospun onto carbon-based material (carbon paper, CP) to obtain an optimized composite anode. The crucial role played by the CP/PEO-NFs anodes was confirmed by the increased proliferation of microorganisms compared to that reached on bare CP anodes, used as a reference material. A parameter named recovered energy (Erec) was introduced to determine the capability of bacteria to oxidize honey and was compared with the Erec obtained when sodium acetate was used as a fuel. CP/PEO-NFs anodes allowed achieving an Erec three times higher than the one reached with a bare carbon-based anode.

Photofabrication of polymeric biomicrofluidics: New insights into material selection.

We propose a versatile method to evaluate the suitability of polymers for the fabrication of microfluidic devices for biomedical applications, based on the concept that the selection and the design of convenient materials should involve different properties depending on the final microfluidic application. Here polymerase chain reaction (PCR) is selected as biological model and target microfluidic reaction. A class of photocured siloxanes is introduced as device building polymers and copolymerization is adopted as strategy to finely tune and optimize the final material properties. All-polymeric flexible devices are easily fabricated exploiting the rapidity of the photopolymerization reaction: they resist to thermal cycles without leakage or de-bonding (i.e., without separation of different chip parts made of the same material bonded together), show very limited water swelling and permeability, are bioinert and prevent the inhibition of the biochemical reaction. PCR is thus successfully conducted in the photocured microfluidic devices made with a specifically designed siloxane copolymer.

P3HT Processing Study for In-Liquid EGOFET Biosensors: Effects of the Solvent and the Surface.

In-liquid biosensing is the new frontier of health and environment monitoring. A growing number of analytes and biomarkers of interest correlated to different diseases have been found, and the miniaturized devices belonging to the class of biosensors represent an accurate and cost-effective solution to obtaining their recognition. In this study, we investigate the effect of the solvent and of the substrate modification on thin films of organic semiconductor Poly(3-hexylthiophene) (P3HT) in order to improve the stability and electrical properties of an Electrolyte Gated Organic Field Effect Transistor (EGOFET) biosensor. The studied surface is the relevant interface between the P3HT and the electrolyte acting as gate dielectric for in-liquid detection of an analyte. Atomic Force Microscopy (AFM) and X-ray Photoelectron Spectroscopy (XPS) characterizations were employed to study the effect of two solvents (toluene and 1,2-dichlorobenzene) and of a commercial adhesion promoter (Ti Prime) on the morphological structure and electronic properties of P3HT film. Combining the results from these surface characterizations with electrical measurements, we investigate the changes on the EGOFET performances and stability in deionized (DI) water with an Ag/AgCl gate electrode.

Scaling Organic Electrochemical Transistors Down to Nanosized Channels.

The perspective of downscaling organic electrochemical transistors (OECTs) in the nanorange is approached by depositing poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) on electrodes with a nanogap designed and fabricated by electromigration induced break junction (EIBJ) technique. The electrical response of the fabricated devices is obtained by acquiring transfer characteristics in order to clarify the specific main characteristics of OECTs with sub-micrometer-sized active channels (nanogap-OECTs). On the basis of their electrical response to different scan times, the nanogap-OECT shows a maximum transconductance unaffected upon changing scan times in the time window from 1 s to 100 µs, meaning that fast varying signals can be easily acquired with unchanged amplifying performance. Hence, the scaling down of the channel size to the nanometer scale leads to a geometrical paradigm that minimizes effects on device response due to the cationic diffusion into the polymeric channel. A comprehensive study of these features is carried out by an electrochemical impedance spectroscopy (EIS) study, complemented by a quantitative analysis made by equivalent circuits. The propagation of a redox front into the polymer bulk due to ionic diffusion also known as the "intercalation pseudocapacitance" is identified as a limiting factor for the transduction dynamics.

Lift-Off Assisted Patterning of Few Layers Graphene.

Graphene and 2D materials have been exploited in a growing number of applications and the quality of the deposited layer has been found to be a critical issue for the functionality of the developed devices. Particularly, Chemical Vapor Deposition (CVD) of high quality graphene should be preserved without defects also in the subsequent processes of transferring and patterning. In this work, a lift-off assisted patterning process of Few Layer Graphene (FLG) has been developed to obtain a significant simplification of the whole transferring method and a conformal growth on micrometre size features. The process is based on the lift-off of the catalyst seed layer prior to the FLG deposition. Starting from a SiO2 finished Silicon substrate, a photolithographic step has been carried out to define the micro patterns, then an evaporation of Pt thin film on Al2O3 adhesion layer has been performed. Subsequently, the Pt/Al2O3 lift-off step has been attained using a dimethyl sulfoxide (DMSO) bath. The FLG was grown directly on the patterned Pt seed layer by Chemical Vapor Deposition (CVD). Raman spectroscopy was applied on the patterned area in order to investigate the quality of the obtained graphene. Following the novel lift-off assisted patterning technique a minimization of the de-wetting phenomenon for temperatures up to 1000 °C was achieved and micropatterns, down to 10 µm, were easily covered with a high quality FLG.