A Rapidly Responsive Sensor for Wireless Detection of Early and Mature Microbial Biofilms.

Biofilm-associated infections, which are able to resist antibiotics, pose a significant challenge in clinical treatments. Such infections have been linked to various medical conditions, including chronic wounds and implant-associated infections, making them a major public-health concern. Early-detection of biofilm formation offers significant advantages in mitigating adverse effects caused by biofilms. In this work, we aim to explore the feasibility of employing a novel wireless sensor for tracking both early-stage and matured-biofilms formed by the medically relevant bacteria Staphylococcus aureus and Pseudomonas aeruginosa. The sensor utilizes electrochemical reduction of an AgCl layer bridging two silver legs made by inkjet-printing, forming a part of near-field-communication tag antenna. The antenna is interfaced with a carbon cloth designed to promote the growth of microorganisms, thereby serving as an electron source for reduction of the resistive AgCl into a highly-conductive Ag bridge. The AgCl-Ag transformation significantly alters the impedance of the antenna, facilitating wireless identification of an endpoint caused by microbial growth. To the best of our knowledge, this study for the first time presents the evidence showcasing that electrons released through the actions of bacteria can be harnessed to convert AgCl to Ag, thus enabling the wireless, battery-less, and chip-less early-detection of biofilm formation.

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.

Glucose-to-Resistor Transduction Integrated into a Radio-Frequency Antenna for Chip-less and Battery-less Wireless Sensing.

To maximize the potential of 5G infrastructure in healthcare, simple integration of biosensors with wireless tag antennas would be beneficial. This work introduces novel glucose-to-resistor transduction, which enables simple, wireless biosensor design. The biosensor was realized on a near-field communication tag antenna, where a sensing bioanode generated electrical current and electroreduced a nonconducting antenna material into an excellent conductor. For this, a part of the antenna was replaced by a Ag nanoparticle layer oxidized to high-resistance AgCl. The bioanode was based on Au nanoparticle-wired glucose dehydrogenase (GDH). The exposure of the cathode-bioanode to glucose solution resulted in GDH-catalyzed oxidation of glucose at the bioanode with a concomitant reduction of AgCl to highly conducting Ag on the cathode. The AgCl-to-Ag conversion strongly affected the impedance of the antenna circuit, allowing wireless detection of glucose. Mimicking the final application, the proposed wireless biosensor was ultimately evaluated through the measurement of glucose in whole blood, showing good agreement with the values obtained with a commercially available glucometer. This work, for the first time, demonstrates that making a part of the antenna from the AgCl layer allows achieving simple, chip-less, and battery-less wireless sensing of enzyme-catalyzed reduction reaction.

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.

Tissue-based biosensor for monitoring the antioxidant effect of orally administered drugs in the intestine.

For a better understanding of the effect of drugs and their interaction with cells and tissues, there is a need for in vitro and ex vivo model systems which enables studying these events. There are several in vitro methods available to evaluate the antioxidant activity; however, these methods do not factor in the complex in vivo physiology. Here we present an intestinal tissue modified oxygen electrode, used for the detection of the antioxidant effect of orally administered drugs in the presence of H2O2. Antioxidants are essential in the defense against oxidative stress, more specifically against reactive oxygen species such as H2O2. Due to the presence of native catalase in the intestine, with the tissue-based biosensor we were able to detect H2O2 in the range between 50 and 500 µM. The reproducibility of the sensor based on the calculated relative standard deviations was 15 ± 6%. We found that the O2 production by catalase from H2O2 was reduced in the presence of a well-known antioxidant, quinol. This indirectly detected antioxidant activity was also observed in the case of orally administered drugs with a reported anti-inflammatory effect such as mesalazine and paracetamol, while no antioxidant activity was recorded with aspirin and metformin.

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.

Peptide Disc Mediated Control of Membrane Protein Orientation in Supported Lipid Bilayers for Surface-Sensitive Investigations.

In vitro characterization of membrane proteins requires experimental approaches providing mimics of the microenvironment that proteins encounter in native membranes. In this context, supported lipid bilayers provide a suitable platform to investigate membrane proteins by a broad range of surface-sensitive techniques such as neutron reflectometry (NR), quartz crystal microbalance with dissipation monitoring (QCM-D), surface plasmon resonance (SPR), atomic force microscopy (AFM), and fluorescence microscopy. Nevertheless, the successful incorporation of membrane proteins in lipid bilayers with sufficiently high concentration and controlled orientation relative to the bilayer remains challenging. We propose the unconventional use of peptide discs made by phospholipids and amphipathic 18A peptides to mediate the formation of supported phospholipid bilayers with two different types of membrane proteins, CorA and tissue factor (TF). The membrane proteins are reconstituted in peptide discs, deposited on a solid surface, and the peptide molecules are then removed with extensive buffer washes. This leaves a lipid bilayer with a relatively high density of membrane proteins on the support surface. As a very important feature, the strategy allows membrane proteins with one large extramembrane domain to be oriented in the bilayer, thus mimicking the in vivo situation. The method is highly versatile, and we show its general applicability by characterizing with the above-mentioned surface-sensitive techniques two different membrane proteins, which were efficiently loaded in the supported bilayers with ∼0.6% mol/mol (protein/lipid) concentration corresponding to 35% v/v for CorA and 8% v/v for TF. Altogether, the peptide disc mediated formation of supported lipid bilayers with membrane proteins represents an attractive strategy for producing samples for structural and functional investigations of membrane proteins and for preparation of suitable platforms for drug testing or biosensor development.

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.

Proteolytic degradation of gelatin-tannic acid multilayers.

HYPOTHESIS: Gelatin is one of the most popular constituents of biodegradable/edible films. Because of its poor mechanical properties, it typically needs to be cross-linked. In this regard, the use of tannic acid has attracted significant interest. Whereas the biodegradability of gelatin is well established, little is known on how different crosslinking methods affect biodegradability. In most cases, the ionic strength at which protein films are grown has a drastic effect on their structure. Thus, it is expected that by controlling the ionic strength during the growth of cross-linked gelatin films it should be possible to tune the access to relevant cleavage sites by proteases and, therefore, their biodegradability. EXPERIMENTS: Gelatin-tannic acid were grown at different ionic strengths by means of the layer-by-layer self-assembly method. The growth of these multilayers and their response to the presence of different proteases were monitored by means of Electrochemical Impedance Spectroscopy and Quartz Crystal Microbalance with Dissipation. FINDINGS: Gelatin-tannic acid multilayers grown at low ionic strength exhibited a swollen structure that allowed easy access to their cleavage sites by proteases. Multilayers formed at physiological ionic strength exhibited a compacter structure, which limited their proteolytic degradation.

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.

Ex-Vivo Force Spectroscopy of Intestinal Mucosa Reveals the Mechanical Properties of Mucus Blankets.

Mucus is the viscous gel that protects mucosal surfaces. It also plays a crucial role in several diseases as well as in mucosal drug delivery. Because of technical limitations, mucus properties have mainly been addressed by in-vitro studies. However, this approach can lead to artifacts as mucus collection can alter its structure. Here we show that by using an implemented atomic force microscope it is possible to measure the interactions between micro-particles and mucus blankets ex-vivo i.e., on fresh excised mucus-covered tissues. By applying this method to study the small intestine, we were able to quantify the stiffness and adhesiveness of its mucus blanket at different pH values. We also demonstrate the ability of mucus blankets to bind and attract particles hundreds of µm away from their surface, and to trap and bury them even if their size is as big as 15 µm.

Design of a potentially prebiotic and responsive encapsulation material for probiotic bacteria based on chitosan and sulfated ß-glucan.

HYPOTHESIS: Chitosan and sulfated oat ß-glucan are materials suitable to create a prebiotic coating for targeted delivery to gastrointestinal system, using the layer by layer technology. EXPERIMENT: Quartz crystal microbalance with dissipation (QCM-D), spectroscopic ellipsometry (SE) and atomic force microscopy (AFM) were used to assess the multilayer formation capacity and characterize the resulting coatings in terms of morphology and material properties such as structure and rigidity. The coating of colloidal materials was proven, specifically on L. acidophilus bacteria as measured by changes in the bacterial suspension zeta potential. Viability of coated cells was shown using plate counting method. The coatings on solid surfaces were examined after exposure to mimics of gastrointestinal fluids and a commercially available ß-glucanase. FINDINGS: Successful build-up of multilayers was confirmed with QCM-D and SE. Zeta potential values proved the coating of cells. There was 2 log CFU/mL decrease after coating cells with four alternating layers of chitosan and sulfated ß-glucan when compared to viability of uncoated cells. The coatings were partially degraded after exposure to simulated intestinal fluid and restructured as a result of ß-glucanase treatment, mimicking enzymes present in the microflora of the human gut, but seemed to resist acidic gastric conditions. Therefore, coatings of chitosan and sulfated ß-glucan can potentially be exploited as carriers for probiotics and delicate nutraceuticals.

Structural and Mechanical Properties of Thin Films of Bovine Submaxillary Mucin versus Porcine Gastric Mucin on a Hydrophobic Surface in Aqueous Solutions.

The structural and mechanical properties of thin films generated from two types of mucins, namely, bovine submaxillary mucin (BSM) and porcine gastric mucin (PGM) in aqueous environment were investigated with several bulk and surface analytical techniques. Both mucins generated hydrated films on hydrophobic polydimethylsiloxane (PDMS) surfaces from spontaneous adsorption arising from their amphiphilic characteristic. However, BSM formed more elastic films than PGM at neutral pH condition. This structural difference was manifested from the initial film formation processes to the responses to shear stresses applied to the films. Acidification of environmental pH led to strengthening the elastic character of BSM films with increased adsorbed mass, whereas an opposite trend was observed for PGM films. We propose that this contrast originates from that negatively charged motifs are present for both the central and terminal regions of BSM molecule, whereas a similar magnitude of negative charges is localized at the termini of PGM molecule. Given that hydrophobic motifs acting as an anchor are also localized in the terminal region, electrostatic repulsion between anchoring units of PGM molecules on a nonpolar PDMS surface leads to weakening of the mechanical integrity of the films.

Interaction of Polyelectrolytes with Salivary Pellicles on Hydroxyapatite Surfaces under Erosive Acidic Conditions.

The modification of acidic beverage formulations with food-approved, nonhazardous substances with antierosive properties has been identified as a key strategy for counteracting the prevalence of dental erosion, i.e., the acid-induced dissolution of hydroxyapatite (HA, the main mineral component of tooth surfaces). While many of such substances have been reported, very little is known on how they interact with teeth and inhibit their acid-induced dissolution. With the aim of filling this gap in knowledge, we have studied under acidic conditions the interaction between two polyelectrolytes of differing ionic character, carboxymethyl cellulose (CMC) and chitosan, and saliva-coated hydroxyapatite, i.e., a model for the outer surface of teeth. These studies were performed by means of ellipsometry, quartz crystal microbalance with dissipation monitoring, and atomic force microscopy. We also studied, by means of pH variations, how dissolution of saliva-coated HA is affected by including these polyelectrolytes in the erosive solutions. Our results confirm that salivary films protect HA from acid-induced dissolution, but only for a limited time. If the acid is modified with CMC, this polyelectrolyte incorporates into the salivary films prolonging in time their protective function. Eventually, the CMC-modified salivary films are removed from the HA surfaces. From this moment, HA is continuously coated with CMC, but this offers only a weak protection against erosion. When the acid is modified with the cationic chitosan, the polyelectrolyte adsorbs on top of the salivary films. Chitosan-modified salivary films are also eventually replaced by bare chitosan films. In this case both coatings offer a similar protection against HA dissolution, which is nevertheless notably higher than that offered by CMC.