Water Sorption and Structural Properties of Human Airway Mucus in Health and Muco-Obstructive Diseases.

Muco-obstructive diseases change airway mucus properties, impairing mucociliary transport and increasing the likelihood of infections. To investigate the sorption properties and nanostructures of mucus in health and disease, we investigated mucus samples from patients and cell cultures (cc) from healthy, chronic obstructive pulmonary disease (COPD), and cystic fibrosis (CF) airways. Atomic force microscopy (AFM) revealed mucin monomers with typical barbell structures, where the globule to spacer volume ratio was the highest for CF mucin. Accordingly, synchrotron small-angle X-ray scattering (SAXS) revealed more pronounced scattering from CF mucin globules and suggested shorter carbohydrate side chains in CF mucin and longer side chains in COPD mucin. Quartz crystal microbalance with dissipation (QCM-D) analysis presented water sorption isotherms of the three types of human airway mucus, where, at high relative humidity, COPD mucus had the highest water content compared to cc-CF and healthy airway mucus (HAM). The higher hydration of the COPD mucus is consistent with the observation of longer side chains of the COPD mucins. At low humidity, no dehydration-induced glass transition was observed in healthy and diseased mucus, suggesting mucus remained in a rubbery state. However, in dialyzed cc-HAM, a sorption-desorption hysteresis (typically observed in the glassy state) appeared, suggesting that small molecules present in mucus suppress the glass transition.

Locating critical events in AFM force measurements by means of one-dimensional convolutional neural networks.

Atomic Force Microscopy (AFM) force measurements are a powerful tool for the nano-scale characterization of surface properties. However, the analysis of force measurements requires several processing steps. One is locating different type of events e.g., contact point, adhesions and indentations. At present, there is a lack of algorithms that can automate this process in a reliable way for different types of samples. Moreover, because of their stochastic nature, the acquisition and analysis of a high number of force measurements is typically required. This can result in these experiments becoming an overwhelming task if their analysis is not automated. Here, we propose a Machine Learning approach, the use of one-dimensional convolutional neural networks, to locate specific events within AFM force measurements. Specifically, we focus on locating the contact point, a critical step for the accurate quantification of mechanical properties as well as long-range interactions. We validate this approach on force measurements obtained both on hard and soft surfaces. This approach, which could be easily used to also locate other events e.g., indentations and adhesions, has the potential to significantly facilitate and automate the analysis of AFM force measurements and, therefore, the use of this technique by a wider community.

MUC5B mucin films under mechanical confinement: A combined neutron reflectometry and atomic force microscopy study.

HYPOTHESIS: Among other functions, mucins hydrate and protect biological interfaces from mechanical challenges. Mucins also attract interest as biocompatible coatings with excellent lubrication performance. Therefore, it is of high interest to understand the structural response of mucin films to mechanical challenges. We hypothesized that this could be done with Neutron Reflectometry using a novel sample environment where mechanical confinement is achieved by inflating a membrane against the films. EXPERIMENTS: Oral MUC5B mucin films were investigated by Force Microscopy/Spectroscopy and Neutron Reflectometry both at solid-liquid interfaces and under mechanical confinement. FINDINGS: NR indicated that MUC5B films were almost completely compressed and dehydrated when confined at 1 bar. This was supported by Force Microscopy/Spectroscopy investigations. Force Spectroscopy also indicated that MUC5B films could withstand mechanical confinement by means of steric interactions for pressures lower than âˆ¼ 0.5 bar i.e., mucins could protect interfaces from mechanical challenges of this magnitude while keeping them hydrated. To investigate mucin films under these pressures by means of the employed sample environment for NR, further technological developments are needed. The most critical would be identifying or developing more flexible membranes that would still meet certain requirements like chemical homogeneity and very low roughness.

Portable Prussian Blue-Based Sensor for Bacterial Detection in Urine.

Bacterial infections can affect the skin, lungs, blood, and brain, and are among the leading causes of mortality globally. Early infection detection is critical in diagnosis and treatment but is a time- and work-consuming process taking several days, creating a hitherto unmet need to develop simple, rapid, and accurate methods for bacterial detection at the point of care. The most frequent type of bacterial infection is infection of the urinary tract. Here, we present a wireless-enabled, portable, potentiometric sensor for E. coli. E. coli was chosen as a model bacterium since it is the most common cause of urinary tract infections. The sensing principle is based on reduction of Prussian blue by the metabolic activity of the bacteria, detected by monitoring the potential of the sensor, transferring the sensor signal via Bluetooth, and recording the output on a laptop or a mobile phone. In sensing of bacteria in an artificial urine medium, E. coli was detected in ~4 h (237 ± 19 min; n = 4) and in less than 0.5 h (21 ± 7 min, n = 3) using initial E. coli concentrations of ~103 and 105 cells mL-1, respectively, which is under or on the limit for classification of a urinary tract infection. Detection of E. coli was also demonstrated in authentic urine samples with bacteria concentration as low as 104 cells mL-1, with a similar response recorded between urine samples collected from different volunteers as well as from morning and afternoon urine samples.

Effect of nonionic and amphoteric surfactants on salivary pellicles reconstituted in vitro.

Surfactants are important components of oral care products. Sodium dodecyl sulfate (SDS) is the most common because of its foaming properties, taste and low cost. However, the use of ionic surfactants, especially SDS, is related to several oral mucosa conditions. Thus, there is a high interest in using non-ionic and amphoteric surfactants as they are less irritant. To better understand the performance of these surfactants in oral care products, we investigated their interaction with salivary pellicles i.e., the proteinaceous films that cover surfaces exposed to saliva. Specifically, we focused on pentaethylene glycol monododecyl ether (C12E5) and cocamidopropyl betaine (CAPB) as model nonionic and amphoteric surfactants respectively, and investigated their interaction with reconstituted salivary pellicles with various surface techniques: Quartz Crystal Microbalance with Dissipation, Ellipsometry, Force Spectroscopy and Neutron Reflectometry. Both C12E5 and CAPB were gentler on pellicles than SDS, removing a lower amount. However, their interaction with pellicles differed. Our work indicates that CAPB would mainly interact with the mucin components of pellicles, leading to collapse and dehydration. In contrast, exposure to C12E5 had a minimal effect on the pellicles, mainly resulting in the replacement/solubilisation of some of the components anchoring pellicles to their substrate.

Enabling autonomous scanning probe microscopy imaging of single molecules with deep learning.

Scanning probe microscopies allow investigating surfaces at the nanoscale, in real space and with unparalleled signal-to-noise ratio. However, these microscopies are not used as much as it would be expected considering their potential. The main limitations preventing a broader use are the need of experienced users, the difficulty in data analysis and the time-consuming nature of experiments that require continuous user supervision. In this work, we addressed the latter and developed an algorithm that controlled the operation of an Atomic Force Microscope (AFM) that, without the need of user intervention, allowed acquiring multiple high-resolution images of different molecules. We used DNA on mica as a model sample to test our control algorithm, which made use of two deep learning techniques that so far have not been used for real time SPM automation. One was an object detector, YOLOv3, which provided the location of molecules in the captured images. The second was a Siamese network that could identify the same molecule in different images. This allowed both performing a series of images on selected molecules while incrementing the resolution, as well as keeping track of molecules already imaged at high resolution, avoiding loops where the same molecule would be imaged an unlimited number of times. Overall, our implementation of deep learning techniques brings SPM a step closer to full autonomous operation.

A comparison between the structures of reconstituted salivary pellicles and oral mucin (MUC5B) films.

HYPOTHESIS: Salivary pellicles i.e., thin films formed upon selective adsorption of saliva, protect oral surfaces against chemical and mechanical insults. Pellicles are also excellent aqueous lubricants. It is generally accepted that reconstituted pellicles have a two-layer structure, where the outer layer is mainly composed of MUC5B mucins. We hypothesized that by comparing the effect of ionic strength on reconstituted pellicles and MUC5B films we could gain further insight into the pellicle structure. EXPERIMENTS: Salivary pellicles and MUC5B films reconstituted on solid surfaces were investigated at different ionic strengths by Force Spectroscopy, Quartz Crystal Microbalance with Dissipation, Null Ellipsometry and Neutron Reflectometry. FINDINGS: Our results support the two-layer structure for reconstituted salivary pellicles. The outer layer swelled when ionic strength decreased, indicating a weak polyelectrolyte behavior. While initially the MUC5B films exhibited a similar tendency, this was followed by a drastic collapse indicating an interaction between exposed hydrophobic domains. This suggests that mucins in the pellicle outer layer form complexes with other salivary components that prevent this interaction. Lowering ionic strength below physiological values also led to a partial removal of the pellicle inner layer. Overall, our results highlight the importance that the interactions of mucins with other pellicle components play on their structure.

Highly Stable Passive Wireless Sensor for Protease Activity Based on Fatty Acid-Coupled Gelatin Composite Films.

Proteases are often used as biomarkers of many pathologies as well as of microbial contamination and infection. Therefore, extensive efforts are devoted to the development of protease sensors. Some applications would benefit from wireless monitoring of proteolytic activity at minimal cost, e.g., sensors embedded in care products like wound dressings and diapers to track wound and urinary infections. Passive (batteryless) and chipless transponders stand out among wireless sensing technologies when low cost is a requirement. Here, we developed and extensively characterized a composite material that is biodegradable but still highly stable in aqueous media, whose proteolytic degradation could be used in these wireless transponders as a transduction mechanism of proteolytic activity. This composite material consisted of a cross-linked gelatin network with incorporated caprylic acid. The digestion of the composite when exposed to proteases results in a change of its resistivity, a quantity that can be wirelessly monitored by coupling the composite to an inductor-capacitor resonator, i.e., an antenna. We experimentally proved this wireless sensor concept by monitoring the presence of a variety of proteases in aqueous media. Moreover, we also showed that detection time follows a relationship with protease concentration, which enables quantification possibilities for practical applications.

Design and use of model membranes to study biomolecular interactions using complementary surface-sensitive techniques.

Cellular membranes are complex structures and simplified analogues in the form of model membranes or biomembranes are used as platforms to understand fundamental properties of the membrane itself as well as interactions with various biomolecules such as drugs, peptides and proteins. Model membranes at the air-liquid and solid-liquid interfaces can be studied using a range of complementary surface-sensitive techniques to give a detailed picture of both the structure and physicochemical properties of the membrane and its resulting interactions. In this review, we will present the main planar model membranes used in the field to date with a focus on monolayers at the air-liquid interface, supported lipid bilayers at the solid-liquid interface and advanced membrane models such as tethered and floating membranes. We will then briefly present the principles as well as the main type of information on molecular interactions at model membranes accessible using a Langmuir trough, quartz crystal microbalance with dissipation monitoring, ellipsometry, atomic force microscopy, Brewster angle microscopy, Infrared spectroscopy, and neutron and X-ray reflectometry. A consistent example for following biomolecular interactions at model membranes is used across many of the techniques in terms of the well-studied antimicrobial peptide Melittin. The overall objective is to establish an understanding of the information accessible from each technique, their respective advantages and limitations, and their complementarity.

Sensing by wireless reading Ag/AgCl redox conversion on RFID tag: universal, battery-less biosensor design.

Massive integration of biosensors into design of Internet-of-Things (IoT) is vital for progress of healthcare. However, the integration of biosensors is challenging due to limited availability of battery-less biosensor designs. In this work, a combination of nanomaterials for wireless sensing of biological redox reactions is described. The design exploits silver nanoparticles (AgNPs) as part of the RFID tag antenna. We demonstrate that a redox enzyme, particularly, horseradish peroxidase (HRP), can convert AgNPs into AgCl in the presence of its substrate, hydrogen peroxide. This strongly changes the impedance of the tag. The presented example exploits gold nanoparticle (AuNP)-assisted electron transfer (ET) between AgNPs and HRP. We show that AuNP is a vital intermediate for establishing rapid ET between the enzyme and AgNPs. As an example, battery-less biosensor-RFID tag designs for H2O2 and glucose are demonstrated. Similar battery-less sensors can be constructed to sense redox reactions catalysed by other oxidoreductase enzymes, their combinations, bacteria or other biological and even non-biological catalysts. In this work, a fast and general route for converting a high number of redox reaction based sensors into battery-less sensor-RFID tags is described.

Effect of Relative Humidity on the Viscoelasticity of Thin Organic Films Studied by Contact Thermal Noise AFM.

Material scientists are in need of experimental techniques that facilitate a quantitative mechanical characterization of mesoscale materials and, therefore, their rational design. An example is that of thin organic films, as their performance often relates to their ability to withstand use without damage. The mechanical characterization of thin films has benefited from the emergence of the atomic force microscope (AFM). In this regard, it is of relevance that most soft materials are not elastic but viscoelastic instead. While most AFM operation modes and analysis procedures are suitable for elasticity studies, the use of AFM for quantitative viscoelastic characterizations is still a challenge. This is now an emerging topic due to recent developments in contact resonance AFM. The aim of this work was to further explore the potential of this technique by investigating its sensitivity to viscoelastic changes induced by environmental parameters, specifically humidity. Here, we show that by means of this experimental approach, it was possible to quantitatively monitor the influence of humidity on the viscoelasticity of two different thin and hydrophobic polyurethane coatings representative of those typically used to protect materials from processes like weathering and wear. The technique was sensitive even to the transition between the antiplasticizing and plasticizing effects of ambient humidity. Moreover, we showed that this was possible without the need of externally exciting the AFM cantilever or the sample, i.e., just by monitoring the Brownian motion of cantilevers, which significantly facilitates the implementation of this technique in any AFM setup.

Humidity-Induced Phase Transitions of Surfactants Embedded in Latex Coatings Can Drastically Alter Their Water Barrier and Mechanical Properties.

Latex coatings are environmentally friendly i.e., they are formed from aqueous polymer dispersions, are cheap to produce and provide exceptional mechanical properties. Therefore, they are ubiquitous and can be found in a wide range of different applications such as paints and varnishes, pressure-sensitive adhesives, textiles, construction materials, paper coatings and inks. However, they also have weaknesses and their surfactant content is among them. Surfactants are often needed to stabilize polymer particles in the aqueous latex dispersions. These surfactants also form part of the coatings formed from these dispersions, and it is well-known that they can lower their performance. This work further explores this aspect and focuses on the role that embedded surfactant domains play in the response of latex coatings to humid environments. For this purpose, we made use of several experimental techniques where humidity control was implemented: quartz crystal microbalance with dissipation, atomic force microscopy and differential scanning calorimetry. By means of this multimethodological approach, we report that surfactants embedded in latex coatings can undergo humidity-induced transitions towards more hydrated and softer phases, and that this results in a drastic decrease of the mechanical and water barrier properties of the whole coatings. Subsequently, this work highlights the potential of taking into account the phase behavior of surfactants when choosing which ones to use in the synthesis of latex dispersions as this would help in predicting their performance under different environmental conditions.