Negligible risk of surface transmission of SARS-CoV-2 in public transportation.

BACKGROUND: Exposure to pathogens in public transport systems is a common means of spreading infection, mainly by inhaling aerosol or droplets from infected individuals. Such particles also contaminate surfaces, creating a potential surface-transmission pathway. METHODS: A fast acoustic biosensor with an antifouling nano-coating was introduced to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on exposed surfaces in the Prague Public Transport System. Samples were measured directly without pre-treatment. Results with the sensor gave excellent agreement with parallel quantitative reverse-transcription polymerase chain reaction (qRT-PCR) measurements on 482 surface samples taken from actively used trams, buses, metro trains and platforms between 7 and 9 April 2021, in the middle of the lineage Alpha SARS-CoV-2 epidemic wave when 1 in 240 people were COVID-19 positive in Prague. RESULTS: Only ten of the 482 surface swabs produced positive results and none of them contained virus particles capable of replication, indicating that positive samples contained inactive virus particles and/or fragments. Measurements of the rate of decay of SARS-CoV-2 on frequently touched surface materials showed that the virus did not remain viable longer than 1-4 h. The rate of inactivation was the fastest on rubber handrails in metro escalators and the slowest on hard-plastic seats, window glasses and stainless-steel grab rails. As a result of this study, Prague Public Transport Systems revised their cleaning protocols and the lengths of parking times during the pandemic. CONCLUSIONS: Our findings suggest that surface transmission played no or negligible role in spreading SARS-CoV-2 in Prague. The results also demonstrate the potential of the new biosensor to serve as a complementary screening tool in epidemic monitoring and prognosis.

Biorecognition antifouling coatings in complex biological fluids: a review of functionalization aspects.

Recent progress in biointerface research has highlighted the role of antifouling functionalizable coatings in the development of advanced biosensors for point-of-care bioanalytical and biomedical applications dealing with real-world complex samples. The resistance to nonspecific adsorption promotes the biorecognition performance and overall increases the reliability and specificity of the analysis. However, the process of modification with biorecognition elements (so-called functionalization) may influence the resulting antifouling properties. The extent of these effects concerning both functionalization procedures potentially changing the surface architecture and properties, and the physicochemical properties of anchored biorecognition elements, remains unclear and has not been summarized in the literature yet. This critical review summarizes these key functionalization aspects with respect to diverse antifouling architectures showing low or ultra-low fouling quantitative characteristics in complex biological media such as bodily fluids or raw food samples. The subsequent discussion focuses on the impact of functionalization on fouling resistance. Furthermore, this review discusses some of the drawbacks of available surface sensitive characterization methods and highlights the importance of suitable assessment of the resistance to fouling.

Functionalized Terpolymer-Brush-Based Biointerface with Improved Antifouling Properties for Ultra-Sensitive Direct Detection of Virus in Crude Clinical Samples.

New analytical techniques that overcome major drawbacks of current routinely used viral infection diagnosis methods, i.e., the long analysis time and laboriousness of real-time reverse-transcription polymerase chain reaction (qRT-PCR) and the insufficient sensitivity of "antigen tests", are urgently needed in the context of SARS-CoV-2 and other highly contagious viruses. Here, we report on an antifouling terpolymer-brush biointerface that enables the rapid and sensitive detection of SARS-CoV-2 in untreated clinical samples. The developed biointerface carries a tailored composition of zwitterionic and non-ionic moieties and allows for the significant improvement of antifouling capabilities when postmodified with biorecognition elements and exposed to complex media. When deployed on a surface of piezoelectric sensor and postmodified with human-cell-expressed antibodies specific to the nucleocapsid (N) protein of SARS-CoV-2, it made possible the quantitative analysis of untreated samples by a direct detection assay format without the need of additional amplification steps. Natively occurring N-protein-vRNA complexes, usually disrupted during the sample pre-treatment steps, were detected in the untreated clinical samples. This biosensor design improved the bioassay sensitivity to a clinically relevant limit of detection of 1.3 × 104 PFU/mL within a detection time of only 20 min. The high specificity toward N-protein-vRNA complexes was validated both by mass spectrometry and qRT-PCR. The performance characteristics were confirmed by qRT-PCR through a comparative study using a set of clinical nasopharyngeal swab samples. We further demonstrate the extraordinary fouling resistance of this biointerface through exposure to other commonly used crude biological samples (including blood plasma, oropharyngeal, stool, and nasopharyngeal swabs), measured via both the surface plasmon resonance and piezoelectric measurements, which highlights the potential to serve as a generic platform for a wide range of biosensing applications.

Atomic force microscopy and surface plasmon resonance for real-time single-cell monitoring of bacteriophage-mediated lysis of bacteria.

The growing incidence of multidrug-resistant bacterial strains presents a major challenge in modern medicine. Antibiotic resistance is often exhibited by Staphylococcus aureus, which causes severe infections in human and animal hosts and leads to significant economic losses. Antimicrobial agents with enzymatic activity (enzybiotics) and phage therapy represent promising and effective alternatives to classic antibiotics. However, new tools are needed to study phage-bacteria interactions and bacterial lysis with high resolution and in real-time. Here, we introduce a method for studying the lysis of S. aureus at the single-cell level in real-time using atomic force microscopy (AFM) in liquid. We demonstrate the ability of the method to monitor the effect of the enzyme lysostaphin on S. aureus and the lytic action of the Podoviridae phage P68. AFM allowed the topographic and biomechanical properties of individual bacterial cells to be monitored at high resolution over the course of their lysis, under near-physiological conditions. Changes in the stiffness of S. aureus cells during lysis were studied by analyzing force-distance curves to determine Young's modulus. This allowed observing a progressive decline in cellular stiffness corresponding to the disintegration of the cell envelope. The AFM experiments were complemented by surface plasmon resonance (SPR) experiments that provided information on the kinetics of phage-bacterium binding and the subsequent lytic processes. This approach forms the foundation of an innovative framework for studying the lysis of individual bacteria that may facilitate the further development of phage therapy.

Functionalizable Antifouling Coatings as Tunable Platforms for the Stress-Driven Manipulation of Living Cell Machinery.

Cells are continuously sensing their microenvironment and subsequently respond to different physicochemical cues by the activation or inhibition of different signaling pathways. To study a very complex cellular response, it is necessary to diminish background environmental influences and highlight the particular event. However, surface-driven nonspecific interactions of the abundant biomolecules from the environment influence the targeted cell response significantly. Yes-associated protein (YAP) translocation may serve as a marker of human hepatocellular carcinoma (Huh7) cell responses to the extracellular matrix and surface-mediated stresses. Here, we propose a platform of tunable functionable antifouling poly(carboxybetain) (pCB)-based brushes to achieve a molecularly clean background for studying arginine, glycine, and aspartic acid (RGD)-induced YAP-connected mechanotransduction. Using two different sets of RGD-functionalized zwitterionic antifouling coatings with varying compositions of the antifouling layer, a clear correlation of YAP distribution with RGD functionalization concentrations was observed. On the other hand, commonly used surface passivation by the oligo(ethylene glycol)-based self-assembled monolayer (SAM) shows no potential to induce dependency of the YAP distribution on RGD concentrations. The results indicate that the antifouling background is a crucial component of surface-based cellular response studies, and pCB-based zwitterionic antifouling brush architectures may serve as a potential next-generation easily functionable surface platform for the monitoring and quantification of cellular processes.

Surface Preconditioning Influences the Antifouling Capabilities of Zwitterionic and Nonionic Polymer Brushes.

Polymer brushes not only represent emerging surface platforms for numerous bioanalytical and biological applications but also create advanced surface-tethered systems to mimic real-life biological processes. In particular, zwitterionic and nonionic polymer brushes have been intensively studied because of their extraordinary resistance to nonspecific adsorption of biomolecules (antifouling characteristics) as well as the ability to be functionalized with bioactive molecules. However, the relation between antifouling behavior in real-world biological media and structural changes of polymer brushes induced by surface preconditioning in different environments remains unexplored. In this work, we use multiple methods to study the structural properties of numerous brushes under variable ionic concentrations and determine the impact of these changes on resistance to fouling from undiluted blood plasma. We describe different mechanisms of swelling, depending on both the polymer brush coating properties and the environmental conditions that affect changes in both hydration levels and thickness. Using both fluorescent and surface plasmon resonance methods, we found that the antifouling behavior of these brushes is strongly dependent on the aforementioned structural changes. Moreover, preconditioning of the brush coatings (incubation at a variable salt concentration or drying) prior to biomolecule interaction may significantly improve the antifouling performance. These results suggest a new simple approach to improve the antifouling behavior of polymer brushes. In addition, the results herein enhance the understanding for improved design of antifouling and bioresponsive brushes employed in biosensor and biomimetic applications.

Modulation of Living Cell Behavior with Ultra-Low Fouling Polymer Brush Interfaces.

Ultra-low fouling and functionalizable coatings represent emerging surface platforms for various analytical and biomedical applications such as those involving examination of cellular interactions in their native environments. Ultra-low fouling surface platforms as advanced interfaces enabling modulation of behavior of living cells via tuning surface physicochemical properties are presented and studied. The state-of-art ultra-low fouling surface-grafted polymer brushes of zwitterionic poly(carboxybetaine acrylamide), nonionic poly(N-(2-hydroxypropyl)methacrylamide), and random copolymers of carboxybetaine methacrylamide (CBMAA) and HPMAA [p(CBMAA-co-HPMAA)] with tunable molar contents of CBMAA and HPMAA are employed. Using a model Huh7 cell line, a systematic study of surface wettability, swelling, and charge effects on the cell growth, shape, and cytoskeleton distribution is performed. This study reveals that ultra-low fouling interfaces with a high content of zwitterionic moieties (>65 mol%) modulate cell behavior in a distinctly different way compared to coatings with a high content of nonionic HPMAA. These differences are attributed mostly to the surface hydration capabilities. The results demonstrate a high potential of carboxybetaine-rich ultra-low fouling surfaces with high hydration capabilities and minimum background signal interferences to create next-generation bioresponsive interfaces for advanced studies of living objects.

Controlling the Transverse Magneto-Optical Kerr Effect in Cr/NiFe Bilayer Thin Films by Changing the Thicknesses of the Cr Layer.

Here, we demonstrate the impact of ferromagnetic layer coating on controlling the magneto-optical response. We found that the transverse magneto-optical Kerr effect (TMOKE) signal and TMOKE hysteresis loops of Ni80Fe20 thin layers coated with a Cr layer show a strong dependence on the thickness of the Cr layer and the incidence angle of the light. The transmission and reflection spectra were measured over a range of incidence angles and with different wavelengths so as to determine the layers' optical parameters and to explain the TMOKE behavior. The generalized magneto-optical and ellipsometry (GMOE) model based on modified Abeles characteristic matrices was used to examine the agreement between the experimental and theoretical results. A comprehensive theoretical and experimental analysis reveals the possibility to create a TMOKE suppression/enhancement coating at specific controllable incidence angles. This has potential for applications in optical microscopy and sensors.

Ultralow-Fouling Behavior of Biorecognition Coatings Based on Carboxy-Functional Brushes of Zwitterionic Homo- and Copolymers in Blood Plasma: Functionalization Matters.

Fouling from complex biological fluids such as blood plasma to biorecognition element (BRE)-functionalized coatings hampers the use of affinity biosensor technologies in medical diagnostics. Here, we report the effects the molecular mechanisms involved in functionalization of low-fouling carboxy-functional coatings have on the BRE capacity and resistance to fouling from blood plasma. The specific mechanisms of EDC/NHS activation of carboxy groups, BRE attachment, and deactivation of residual activated groups on recently developed ultra-low-fouling carboxybetaine polymer and copolymer brushes (pCB) as well as conventional carboxy-terminated oligo(ethylene glycol)-based alkanethiolate self-assembled monolayers (OEG-SAMs) are studied using the polarization modulation infrared reflection/absorption spectroscopy, X-ray photoelectron spectroscopy, and surface plasmon resonance methods. It is shown that the fouling resistance of BRE-functionalized pCB coatings is strongly influenced by a deactivation method affecting the ultra-low-fouling molecular structure of the brush and surface charges. It is revealed that, in contrast to free carboxy-group-terminated OEG-SAMs, only a partial deactivation of EDC/NHS-activated zwitterionic carboxy groups by spontaneous hydrolysis is possible in the pCB brushes. The fouling resistance of activated/BRE-functionalized pCB is shown to be recovered only by covalent attachment of amino acid deactivation agents to residual activated carboxy groups of pCB. The developed deactivation procedure is further combined with ultra-low-fouling brushes of random copolymer carboxybetaine methacrylamide (CBMAA) and N-(2-hydroxypropyl) methacrylamide (HPMAA) with optimized CBMAA content (15%) providing a BRE-functionalized coating with superior fouling resistance over various carboxy-functional low-fouling coatings including homopolymer pCB brushes and OEG-SAMs. The biorecognition capabilities of pHPMAA-CBMAA(15%) are demonstrated via the sensitive label-free detection of a microRNA cancer biomarker (miR-16) in blood plasma.

Copolymer Brush-Based Ultralow-Fouling Biorecognition Surface Platform for Food Safety.

Functional polymer coatings that combine the ability to resist nonspecific fouling from complex media with high biorecognition element (BRE) immobilization capacity represent an emerging class of new functional materials for a number of bioanalytical and biosensor technologies for medical diagnostics, security, and food safety. Here, we report on a random copolymer brush surface - poly(CBMAA-ran-HPMAA) - providing high BRE immobilization capacity while simultaneously exhibiting ultralow-fouling behavior in complex food media. We demonstrate that both the functionalization and fouling resistance capabilities of such copolymer brushes can be tuned by changing the surface contents of the two monomer units: nonionic N-(2-hydroxypropyl) methacrylamide (HPMAA) and carboxy-functional zwitterionic carboxybetaine methacrylamide (CBMAA). It is demonstrated that the resistance to fouling decreases with the surface content of CBMAA; poly(CBMAA-ran-HPMAA) brushes with CBMAA molar content up to 15 mol % maintain excellent resistance to fouling from a variety of homogenized foods (hamburger, cucumber, milk, and lettuce) even after covalent attachment of BREs to carboxy groups of CBMAA. The poly(CBMAA 15 mol %-ran-HPMAA) brushes functionalized with antibodies are demonstrated to exhibit fouling resistance from food samples by up to 3 orders of magnitude better when compared with the widely used low-fouling carboxy-functional oligo(ethylene glycol) (OEG)-based alkanethiolate self-assembled monolayers (AT SAMs) and, furthermore, by up to 2 orders of magnitude better when compared with the most successful ultralow-fouling biorecognition coatings - poly(carboxybetaine acrylamide), poly(CBAA). When model SPR detections of food-borne bacterial pathogens in homogenized foods are used, it is also demonstrated that the antibody-functionalized poly(CBMAA 15 mol %-ran-HPMAA) brush exhibits superior biorecognition properties over the poly(CBAA).

Low-fouling surface plasmon resonance biosensor for multi-step detection of foodborne bacterial pathogens in complex food samples.

Recent outbreaks of foodborne illnesses have shown that foodborne bacterial pathogens present a significant threat to public health, resulting in an increased need for technologies capable of fast and reliable screening of food commodities. The optimal method of pathogen detection in foods should: (i) be rapid, specific, and sensitive; (ii) require minimum sample preparation; and (iii) be robust and cost-effective, thus enabling use in the field. Here we report the use of a SPR biosensor based on ultra-low fouling and functionalizable poly(carboxybetaine acrylamide) (pCBAA) brushes for the rapid and sensitive detection of bacterial pathogens in crude food samples utilizing a three-step detection assay. We studied both the surface resistance to fouling and the functional capabilities of these brushes with respect to each step of the assay, namely: (I) incubation of the sensor with crude food samples, resulting in the capture of bacteria by antibodies immobilized to the pCBAA coating, (II) binding of secondary biotinylated antibody (Ab2) to previously captured bacteria, and (III) binding of streptavidin-coated gold nanoparticles to the biotinylated Ab2 in order to enhance the sensor response. We also investigated the effects of the brush thickness on the biorecognition capabilities of the gold-grafted functionalized pCBAA coatings. We demonstrate that pCBAA-compared to standard low-fouling OEG-based alkanethiolate self-assemabled monolayers-exhibits superior surface resistance regarding both fouling from complex food samples as well as the non-specific binding of S-AuNPs. We further demonstrate that a SPR biosensor based on a pCBAA brush with a thickness as low as 20 nm was capable of detecting E. coli O157:H7 and Salmonella sp. in complex hamburger and cucumber samples with extraordinary sensitivity and specificity. The limits of detection for the two bacteria in cucumber and hamburger extracts were determined to be 57 CFU/mL and 17 CFU/mL for E. coli and 7.4 × 10(3) CFU/mL and 11.7 × 10(3)CFU/mL for Salmonella sp., respectively. In addition, we demonstrate the simultaneous detection of E. coli and Salmonella sp. in hamburger sample using a multichannel SPR biosensor having appropriate functional coatings.

Rapid and sensitive detection of multiple microRNAs in cell lysate by low-fouling surface plasmon resonance biosensor.

We report an ultra-low fouling surface plasmon resonance imaging (SPRi) biosensor for the rapid simultaneous detection of multiple miRNAs in erythrocyte lysate (EL) at subpicomolar levels without need of RNA extraction. The SPRi chips were coated with ultra-low fouling functionalizable poly(carboxybetaine acrylamide) (pCBAA) brushes having optimized thicknesses and directly functionalized with amino-modified oligonucleotide probes. We have characterized the effect of the brush thickness on the probe loading capacity: a loading capacity of ~9.8×10(12) probes/cm(2) was achieved for pCBAA having a thickness of ~40 nm. The probe-functionalized sensor also exhibited a high resistance to fouling from ~90% EL samples (<2 ng/cm(2)). A two-step detection assay was employed for multiplexed miRNA detection in EL. Specifically, the assay consisted of (i) a sandwich-type hybridization of the probe-functionalized pCBAA with target miRNA in EL (bound to biotinylated oligonucleotides) and (ii) the capture of streptavidin-functionalized gold nanoparticles to the aforementioned biotinylated probes. We have demonstrated that this approach enables the detection of miRNAs in EL at concentrations as low as 0.5 pM. Finally, we have confirmed the detection of four endogenous miRNAs representing a set of potential miRNA biomarkers of myelodysplastic syndrome (MDS) in clinical EL samples (miR-16, miR-181, miR-34a, and miR-125b). The results revealed significantly higher levels of miR-16 in all the clinical EL samples compared to the other measured miRNAs.