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.

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.

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.

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.

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.

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.

Anti-adherence and bactericidal activity of sphingolipids against Streptococcus mutans.

This study evaluated the anti-biofilm activity of sphingosine, phytosphingosine (PHS), and sphinganine for: (i) anti-adherence activity on hydroxyapatite (HA) surfaces; and (ii) bactericidal activity on different Streptococcus mutans phenotypes (i.e. planktonic cells and cells from a disrupted biofilm). For this, HA discs treated with sphingolipids were incubated with S. mutans and the number of adherent cells was evaluated by both culture and confocal microscopy. Sphinganine strongly inhibited bacterial adherence by 1000-fold compared with an untreated surface. Phytosphingosine and sphingosine inhibited bacterial adherence by eight- and five-fold, respectively, compared with an untreated surface. On saliva-coated HA, sphinganine and PHS inhibited bacterial adherence by 10-fold. Bactericidal activity of sphingolipids was evaluated by culture. For biofilms, the strongest bactericidal activity was exhibited by sphingosine compared with PHS and sphinganine. At a concentration of 12.5 µg ml(-1) , PHS and sphingosine were profoundly effective against planktonic and disrupted biofilms; and sphinganine reduced the number of cells in planktonic form by 100-fold and those derived from a disrupted biofilm by 1000-fold. Atomic force microscopy studies suggested that mechanical stability does not appear to be a factor relevant for anti-fouling activity. The results suggest that sphingolipids may be used to control oral biofilms, especially those loaded with S. mutans.

Sphingoid bases inhibit acid-induced demineralization of hydroxyapatite.

Calcium hydroxyapatite (HAp), the main constituent of dental enamel, is inherently susceptible to the etching and dissolving action of acids, resulting in tooth decay such as dental caries and dental erosion. Since the prevalence of erosive wear is gradually increasing, there is urgent need for agents that protect the enamel against erosive attacks. In the present study we studied in vitro the anti-erosive effects of a number of sphingolipids and sphingoid bases, which form the backbone of sphingolipids. Pretreatment of HAp discs with sphingosine, phytosphingosine (PHS), PHS phosphate and sphinganine significantly protected these against acid-induced demineralization by 80 ± 17%, 78 ± 17%, 78 ± 7% and 81 ± 8%, respectively (p < 0.001). On the other hand, sphingomyelin, acetyl PHS, octanoyl PHS and stearoyl PHS had no anti-erosive effects. Atomic force measurement revealed that HAp discs treated with PHS were almost completely and homogeneously covered by patches of PHS. This suggests that PHS and other sphingoid bases form layers on the surface of HAp, which act as diffusion barriers against H(+) ions. In principle, these anti-erosive properties make PHS and related sphingosines promising and attractive candidates as ingredients in oral care products.

Interfacial behavior and activity of laccase and bilirubin oxidase on bare gold surfaces.

Two blue multicopper oxidases (MCOs) (viz. Trametes hirsuta laccase (ThLc) and Myrothecium verrucaria bilirubin oxidase (MvBOx)) were immobilized on bare polycrystalline gold (Au) surfaces by direct adsorption from both dilute and concentrated enzyme solutions. The adsorption was studied in situ by means of null ellipsometry. Moreover, both enzyme-modified and bare Au electrodes were investigated in detail by atomic force microscopy (AFM) as well as electrochemically. When adsorbed from dilute solutions (0.125 and 0.25 mg mL⁻¹ in the cases of ThLc and MvBOx, respectively), the amounts of enzyme per unit area were determined to be ca. 1.7 and 4.8 pmol cm⁻², whereas the protein film thicknesses were determined to be 29 and 30 Å for ThLc and MvBOx, respectively. A well-pronounced bioelectrocatalytic reduction of molecular oxygen (O2) was observed on MvBOx/Au biocathodes, whereas this was not the case for ThLc-modified Au electrodes (i.e., adsorbed ThLc was catalytically inactive). The initially observed apparent k(cat)(app) values for adsorbed MvBOx and the enzyme in solution were found to be very close to each other (viz. 54 and 58 s⁻¹, respectively (pH 7.4, 25 °C)). However, after 3 h of operation of MvBOx/Au biocathodes, kcatapp dropped to 23 s⁻¹. On the basis of the experimental results, conformational changes of the enzymes (in all likelihood, their flattening on the Au surface) were suggested to explain the deactivation of MCOs on the bare Au electrodes.

Influence of substratum hydrophobicity on salivary pellicles: organization or composition?

Different physico-chemical properties (eg adsorption kinetics, thickness, viscoelasticity, and mechanical stability) of adsorbed salivary pellicles depend on different factors, including the properties (eg charge, roughness, wettability, and surface chemistry) of the substratum. Whether these differences in the physico-chemical properties are a result of differences in the composition or in the organization of the pellicles is not known. In this work, the influence of substratum wettability on the composition of the pellicle was studied. For this purpose, pellicles eluted from substrata of different but well-characterized wettabilities were examined by means of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The results showed that substratum hydrophobicity did not have a major impact on pellicle composition. In all substrata, the major pellicle components were found to be cystatins, amylases and large glycoproteins, presumably mucins. In turn, interpretation of previously reported data based on the present results suggests that variations in substratum wettability mostly affect the organization of the pellicle components.

The biological response to three different nanostructures applied on smooth implant surfaces.

OBJECTIVE: To evaluate the biological effects of three calcium phosphate (CaP) coatings with nanostructures on relatively smooth implant surfaces. MATERIAL AND METHODS: Stable CaP nanoparticle suspensions of different particle sizes and structures were coated onto implants by immersion and subsequent heat treatment. An uncoated implant was used as the control. After topographical and chemical characterizations, implants were randomly inserted into rabbit tibiae for removal torque (RTQ) testing. To confirm the biological reaction, implants were placed in the bilateral femurs of three rabbits. RESULTS: The topographical characterization showed that each surface had different nanostructural characteristics and X-ray photon spectroscopy showed various CaP compositions. The control and test groups had different nanotopographies; however, the differences among the test groups were only significant for Surfaces B and C and the rest were insignificant. The RTQ tests showed significantly higher values in two test groups (Surface A and Surface C). Histologically, no adverse effects were seen in any group. Histomorphometrical evaluation showed comparable or better osseointegration along the implant threads in the test groups. CONCLUSION: The three different CaP coatings with nanostructures on the implant surfaces had enhancing effects on osseointegration. Along with the surface nanotopography, the CaP chemistry might have influenced the biological outcomes.

Adsorption of HSA, IgG and laminin-1 on model hydroxyapatite surfaces--effects of surface characteristics.

Ellipsometry and mechanically assisted sodium dodecyl sulphate elution was utilized to study the adsorption of human serum albumin (HSA), human immunoglobulin G (IgG), and laminin-1, as well as competitive adsorption from a mixture of these proteins on spin-coated and sintered hydroxyapatite (HA) surfaces, respectively. The HA surfaces were characterized with respect to wettability and roughness by means of water contact angles and atomic force microscopy, respectively. Both surface types were hydrophilic, and the average roughness (Sa) and surface enlargement (Sdr) were lower for the sintered compared to the spin-coated HA surfaces. The adsorbed amounts on the sintered HA increased as follows: HSA < laminin-1 < IgG < the protein mixture. For the competitive adsorption experiments, the adsorbed fractions increased accordingly: HSA < laminin-1 < IgG on both types of HA substratum. However, a higher relative amount of HSA and laminin-1 and a lower relative amount of IgG was found on the spin-coated surfaces compared to the sintered surfaces. The effects observed could be ascribed to differences in surface roughness and chemical composition between the two types of HA substratum, and could have an influence on selection of future implant surface coatings.

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.

Friction force spectroscopy as a tool to study the strength and lateral diffusion of protein layers.

We present a method to study the strength of layers of biological molecules in liquid medium. The method is based on the Friction Force Spectroscopy operation mode of the Atomic Force Microscope. It works by scratching the sample surface at different applied loads while registering the evolution of the sample topography and of the friction between probe and sample. Results are presented for BSA and ß-casein monolayers on hydrophobic surfaces. We show how the simultaneous monitoring of topography and friction allows detecting differences not only between the strength of both types of layers, but also between the lateral diffusion of the proteins within these layers. Specifically, ß-casein is shown to form stronger layers than BSA. The yield strengths calculated for both of these systems are in the range 50-70 MPa. Moreover, while no lateral diffusion is observed for BSA, we show that ß-casein diffuses along the hydrophobic substrates at a rate higher than the scan velocity of the tip (16 µm s(-1) in our case).

Friction force spectroscopy as a tool to study the strength and structure of salivary films.

In this work, we employ atomic force microscopy based friction force spectroscopy to study the strength and structure of salivary films. Specifically, films formed on model hydrophobic (methylated silica) and hydrophilic (clean silica) substrata have been studied in water at pHs in the range 3.3-7. Results reveal that films formed on both types of substrata can be described in terms of two different fractions, with only one of them being able to diffuse along the underlying substrata. We also show how the protective function of the films is reduced when the pH of the surrounding medium is lowered. Specifically, lowering of pH causes desorption of some components of the films formed on hydrophobic methylated surfaces, leading to weaker layers. In contrast, at low pHs, saliva no longer forms a layer on hydrophilic silica surfaces. Instead, an inhomogeneous distribution of amorphous aggregates is observed. Our data also suggest that hydrophobic materials in the oral cavity might be more easily cleaned from adsorbed salivary films. Finally, reproducible differences are observed in results from experiments on films from different individuals, validating the technique as a tool for clinical diagnosis of the resistance to erosion of salivary films.

Friction force spectroscopy of ß- and κ-casein monolayers.

Friction force spectroscopy (FFS) has been applied to study the tribological properties of ß- and κ-casein layers on hydrophobic substrates in aqueous solutions. Nanometer-sized imaging tips were employed. This allowed exerting and determining the high pressures needed to remove the layers and registering the topographic evolution during this process. Both ß- and κ-casein layers showed similar and not particularly high initial frictional responses (friction coefficient of ∼1 when measured with a silicon nitride tip). The pressures needed to remove the layers were of the same order of magnitude for both proteins, ∼10(8) Pa, but slightly higher for those composed of ß-casein. The technique has also shown to be useful in studying the two-dimensional lateral diffusion of the proteins and the wear on the layers they form.

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.