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Functionalized nanoparticles (NPs) are widely used in targeted drug delivery and biomedical imaging due to their penetration into living cells. The outer coating of most cells is a sugar-rich layer of the cellular glycocalyx, presumably playing an important part in any uptake processes. However, the exact role of the cellular glycocalyx in NP uptake is still uncovered. Here, we in situ monitored the cellular uptake of gold NPs─functionalized with positively charged alkaline thiol (TMA)─into adhered cancer cells with or without preliminary glycocalyx digestion. Proteoglycan (PG) components of the glycocalyx were treated by the chondroitinase ABC enzyme. It acts on chondroitin 4-sulfate, chondroitin 6-sulfate, and dermatan sulfate and slowly on hyaluronate. The uptake measurements of HeLa cells were performed by applying a high-throughput label-free optical biosensor based on resonant waveguide gratings. The positively charged gold NPs were used with different sizes [

The high throughput, cost effective and sensitive quantification of cell adhesion strength at the single-cell level is still a challenging task. The adhesion force between tissue cells and their environment is crucial in all multicellular organisms. Integrins transmit force between the intracellular cytoskeleton and the extracellular matrix. This force is not only a mechanical interaction but a way of signal transduction as well. For instance, adhesion-dependent cells switch to an apoptotic mode in the lack of adhesion forces. Adhesion of tumor cells is a potential therapeutic target, as it is actively modulated during tissue invasion and cell release to the bloodstream resulting in metastasis. We investigated the integrin-mediated adhesion between cancer cells and their RGD (Arg-Gly-Asp) motif displaying biomimetic substratum using the HeLa cell line transfected by the Fucci fluorescent cell cycle reporter construct. We employed a computer-controlled micropipette and a high spatial resolution label-free resonant waveguide grating-based optical sensor calibrated to adhesion force and energy at the single-cell level. We found that the overall adhesion strength of single cancer cells is approximately constant in all phases except the mitotic (M) phase with a significantly lower adhesion. Single-cell evanescent field based biosensor measurements revealed that at the mitotic phase the cell material mass per unit area inside the cell-substratum contact zone is significantly less, too. Importantly, the weaker mitotic adhesion is not simply a direct consequence of the measured smaller contact area. Our results highlight these differences in the mitotic reticular adhesions and confirm that cell adhesion is a promising target of selective cancer drugs as the vast majority of normal, differentiated tissue cells do not enter the M phase and do not divide.

The beat in physical systems is a transparent and well-understood phenomenon. It may occur in forced oscillatory systems and as a result of the interference of two waves of slightly different frequencies. However, in chemical systems, the realization of the latter type of the beat phenomenon has been lacking. Here we show that a periodic titration of acid and alkaline solutions with each other using programmable syringe pumps in a continuous stirred-tank reactor exhibits the beat phenomenon in the temporal pH oscillation pattern if the time periods of sinusoidal inflow rates of the reagents are slightly different. Interestingly, the frequency of the chemical beat pattern follows the well-known relationship from physics, namely the frequency of the beat is equal to the absolute value of the difference of the two wave frequencies. Based on our strategy, we can design and engineer non-autonomous pH oscillatory systems, in which the characteristics of the temporal oscillations (amplitude, time period) can easily and precisely be controlled by the experimental conditions such as the inflow rates and feed concentrations. The demonstrated phenomena can be exploited in practical applications, we use the non-autonomous pH oscillators to drive the reversible assembly and disassembly of pH-sensitive building blocks (oleic acid and gold nanoparticles), both highly relevant in nanotechnology and biomedical applications.

Single-cell adhesion plays an essential role in biological and biomedical sciences, but its precise measurement for a large number of cells is still a challenging task. At present, typical force measuring techniques usually offer low throughput, a few cells per day, and therefore are unable to uncover phenomena emerging at the population level. In this work, robotic fluidic force microscopy (FluidFM) was utilized to measure the adhesion parameters of cells in a high-throughput manner to study their population distributions in-depth. The investigated cell type was the genetically engineered HeLa Fucci construct with cell cycle-dependent expression of fluorescent proteins. This feature, combined with the high-throughput measurement made it possible for the first time to characterize the single-cell adhesion distributions at various stages of the cell cycle. It was found that parameters such as single-cell adhesion force and energy follow a lognormal population distribution. Therefore, conclusions based on adhesion data of a low number of cells or treating the population as normally distributed can be misleading. Moreover, we found that the cell area was significantly the smallest, and the area normalized maximal adhesion force was significantly the largest for the colorless cells (the mitotic (M) and early G1 phases). Notably, the parameter characterizing the elongation of the cells until the maximum level of force between the cell and its substratum was also dependent on the cell cycle, which quantity was the smallest for the colorless cells. A novel parameter, named the spring coefficient of the cell, was introduced as the fraction of maximal adhesion force and maximal cell elongation during the mechanical detachment, which was found to be significantly the largest for the colorless cells. Cells in the M phase adhere in atypical way, with so-called reticular adhesions, which are different from canonical focal adhesions. We first revealed that reticular adhesion can exert a higher force per unit area than canonical focal adhesions, and cells in this phase are significantly stiffer. The possible biological consequences of these findings were also discussed, together with the practical relevance of the observed population-level adhesion phenomena.

Reliable measurement of the binding kinetics of low molecular weight analytes to their targets is still a challenging task. Often, the introduction of labels is simply impossible in such measurements, and the application of label-free methods is the only reliable choice. By measuring the binding kinetics of Ni(II) ions to genetically modified flagellin layers, we demonstrate that: (1) Grating-Coupled Interferometry (GCI) is well suited to resolve the binding of ions, even at very low protein immobilization levels; (2) it supplies high quality kinetic data from which the number and strength of available binding sites can be determined, and (3) the rate constants of the binding events can also be obtained with high accuracy. Experiments were performed using a flagellin variant incorporating the C-terminal domain of the nickel-responsive transcription factor NikR. GCI results were compared to affinity data from titration calorimetry. We found that besides the low-affinity binding sites characterized by a micromolar dissociation constant (Kd), tetrameric FliC-NikRC molecules possess high-affinity binding sites with Kd values in the nanomolar range. GCI enabled us to obtain real-time kinetic data for the specific binding of an analyte with molar mass as low as 59 Da, even at signals lower than 1 pg/mm2.

Resonance, beats, and synchronization are general and fundamental phenomena in physics. Their existence and their in-depth understanding in physical systems have led to several applications and technological developments shaping our world today. Here we show the existence of chemical resonance, chemical beats, and frequency locking phenomena in periodically forced pH oscillatory systems (sulfite-hydrogen peroxide and sulfite-formaldehyde-gluconolactone pH oscillatory systems). Periodic forcing was realized by a superimposed sinusoidal modulation on the inflow rates of the reagents in the continuous-flow stirred tank reactor. The dependence of the time period of beats follows the relation known from classical physics for forced physical oscillators. Our developed numerical model describes qualitatively the resonance and beat phenomena experimentally revealed. Application of periodic forcing in autonomously oscillating systems can provide new types of oscillators with a controllable frequency and new insight into controlling irregular chemical oscillation regimes.

Tissue-on-a-chip technologies are more and more important in the investigation of cellular function and in the development of novel drugs by allowing the direct screening of substances on human cells. Constituting the inner lining of vessel walls, endothelial cells are the key players in various physiological processes, moreover, they are the first to be exposed to most drugs currently used. However, to date, there is still no appropriate technology for the label-free, real-time and high-throughput monitoring of endothelial function. To this end, we developed an optical biosensor-based endothelial label-free biochip (EnLaB) assay that meets all the above requirements. Using our EnLaB platform, we screened a set of plasma serine proteases as possible endothelial cell activators, and first identified the endothelial cell activating function of three important serine proteases – namely kallikrein, C1r and mannan-binding lectin-associated serine-protease 2 (MASP-2) – and verified these results in well-established functional assays. EnLaB proved to be an effective tool for revealing novel cellular mechanisms as well as for the high-throughput screening of various compounds on endothelial cells.

Single-cell adhesion force plays a crucial role in biological sciences, however its in-depth investigation is hindered by the extremely low throughput and the lack of temporal resolution of present techniques. While atomic force microcopy (AFM) based methods are capable of directly measuring the detachment force values between individual cells and a substrate, their throughput is limited to few cells per day, and cannot provide the kinetic evaluation of the adhesion force over the timescale of several hours. In this study a high spatial and temporal resolution resonant waveguide grating based label-free optical biosensor was combined with robotic fluidic force microscopy to monitor the adhesion of living cancer cells. In contrast to traditional fluidic force microscopy methods with a manipulation range in the order of 300–400 micrometers, the robotic device employed here can address single cells over mm-cm scale areas. This feature significantly increased measurement throughput, and opened the way to combine the technology with the employed microplate-based, large area biosensor. After calibrating the biosensor signals with the direct force measuring technology on 30 individual cells, the kinetic evaluation of the adhesion force and energy of large cell populations was performed for the first time. We concluded that the distribution of the single-cell adhesion force and energy can be fitted by log-normal functions as cells are spreading on the surface and revealed the dynamic changes in these distributions. The present methodology opens the way for the quantitative assessment of the kinetics of single-cell adhesion force and energy with an unprecedented throughput and time resolution, in a completely non-invasive manner.

Novel biosensors already provide a fast way to detect the adhesion of whole bacteria (or parts of them), biofilm formation, and the effect of antibiotics. Moreover, the detection sensitivities of recent sensor technologies are large enough to investigate molecular-scale biological processes. Usually, these measurements can be performed in real time without using labeling. Despite these excellent capabilities summarized in the present work, the application of novel, label-free sensor technologies in basic biological research is still rare; the literature is dominated by heuristic work, mostly monitoring the presence and amount of a given analyte. The aims of this review are (i) to give an overview of the present status of label-free biosensors in bacteria monitoring, and (ii) to summarize potential novel directions with biological relevancies to initiate future development. Optical, mechanical, and electrical sensing technologies are all discussed with their detailed capabilities in bacteria monitoring. In order to review potential future applications of the outlined techniques in bacteria research, we summarize the most important kinetic processes relevant to the adhesion and survival of bacterial cells. These processes are potential targets of kinetic investigations employing modern label-free technologies in order to reveal new fundamental aspects. Resistance to antibacterials and to other antimicrobial agents, the most important biological mechanisms in bacterial adhesion and strategies to control adhesion, as well as bacteria-mammalian host cell interactions are all discussed with key relevancies to the future development and applications of biosensors.

Recognition of the important role of transposable elements (TEs) in eukaryotic genomes quickly led to a burgeoning literature modeling and estimating the effects of selection on TEs. Much of the empirical work on selection has focused on analyzing the site frequency spectrum (SFS) of TEs. But TEs differ from standard evolutionary models in a number of ways that can impact the power and interpretation of the SFS. For example, rather than mutating under a clock-like model, transposition often occurs in bursts which can inflate particular frequency categories compared to expectations under a standard neutral model. If a TE burst has been recent, the excess of low frequency polymorphisms can mimic the effect of purifying selection. Here, we investigate how transposition bursts affect the frequency distribution of TEs and the correlation between age and allele frequency. Using information on the TE age distribution, we propose an age-adjusted site frequency spectrum to compare TEs and neutral polymorphisms to more effectively evaluate whether TEs are under selective constraints. We show that our approach can minimize instances of false inference of selective constraint, but also allows for a correct identification of even weak selection affecting TEs which experienced a transposition burst and is robust to at least simple demographic changes. The results presented here will help researchers working on TEs to more reliably identify the effects of selection on TEs without having to rely on the assumption of a constant transposition rate.

The invasiveness of cancer cells describes the metastasizing capability of a primary tumor. The straightforward detection and quantification of cancer cell invasion are important to predict the survival rate of a cancer patient and to test how anti-cancer compounds influence cancer progression. Digital holographic microscopy based M4 Holomonitor (HM) is a technique that allows the label-free monitoring of cell morphological and kinetical parameters in real-time. Here, a fully confluent epithelial monolayer derived from the African green monkey kidney (Vero) on a gelatin-coated surface was established, then HeLa cells were seeded on top of the monolayer, and their behavior was monitored for 24 h using HM. Several cancer cells showing invasiveness were detected during this period, while other HeLa cells did not show any signs of aggressivity. It was demonstrated that the invasion of single cancer cells is soundly observable and also quantifiable through monitoring parameters such as phase shift, optical volume, area, and motility, which parameters can easily be obtained and processed automatically. Based on the experimental data, the invasion speed of cancer cells entering the epithelial layer can be defined as the shrinking of detected single-cell volume per unit time. The invasion speed and its correlation with cell migration parameters were analyzed in depth. A clear linear relationship between migration and invasion speed was found, cancer cells with stronger migration have slower invasion speed. These results not only describe the effect of how cancer cells invade the underlying monolayer in contrast to non-invasive HeLa cells, but could help in future research to optimize drugs affecting cell invasibility in a fully automated, label-free and high-throughput manner.

Cytotoxic effects of the market leading broad-spectrum, synthetic herbicide product Roundup Classic, its active ingredient glyphosate (in a form of its isopropylamine (IPA) salt) and its formulating surfactant polyethoxylated tallowamine (POE-15) were determined on two murine cell lines, a neuroectodermal stem cell-like (NE-4C) and a high alkaline phosphatase activity osteoblastic cell line (MC3T3-E1). Cytotoxicity, genotoxicity, effects on cell viability and cell cycles were examined in five flow cytometry tests, the two former of which were compared by the enzymatic-assay and the alkaline single cell gel electrophoresis (Comet) assay. All of the tests indicated the NE-4C cells being more sensitive, than the MC3T3-E1 cell line to the treatments with the target compounds. Higher sensitivity differences were detected in the viability test by flow cytometry (7-9-fold), than by the MTT assay (1.5-3-fold); in the genotoxicity test by the Comet assay (3.5-403-fold), than by the DNA-damage test (9.3-158-fold); and in the apoptosis test by the Annexin V dead cell kit (1.1-12.7-fold), than by the Caspase 3/7 kit (1-6.5-fold). Cell cycle assays indicated high count of cells (~70%) in the G0/G1 phase for MC3T3-E1 cells, than in NE-4C cell (~40%) after 24 h. The order of the inhibitory potency of the target substances has unequivocally been POE-15 Roundup Classic glyphosate IPA salt.

Plants and fungi can be used for medical applications because of their accumulation of special bioactive metabolites. These substances might be beneficial to human health, exerting also anti-inflammatory and anticancer (antiproliferative) effects. We propose that they are mediated by influencing cellular adhesion and migration via various signaling pathways and by directly inactivating key cell adhesion surface receptor sites. The evidence for this proposition is reviewed (by summarizing the natural metabolites and their effects influencing cellular adhesion and migration), along with the classical measuring techniques used to gain such evidence. We systematize existing knowledge concerning the mechanisms of how natural metabolites affect adhesion and movement, and their role in gene expression as well. We conclude by highlighting the possibilities to screen natural compounds faster and more easily by applying new label-free methods, which also enable a far greater degree of quantification than the conventional methods used hitherto. We have systematically classified recent studies regarding the effects of natural compounds on cellular adhesion and movement, characterizing the active substances according to their organismal origin (plants, animals or fungi). Finally, we also summarize the results of recent studies and experiments on SARS-CoV-2 treatments by natural extracts affecting mainly the adhesion and entry of the virus.

Clonal complex 398 (CC398) livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) has been reported worldwide in a variety of food-animal species. Although CC398 is synonymous with LA-MRSA, community-associated MRSA (CA-MRSA) variants have emerged, including the Panton-Valentine leukocidin (PVL)-positive ST398-V and ST398 single-locus variant ST1232-V, and the PVL-negative ST398-V clones. Using comparative genomic analysis, we determined whether ten CC398 MRSA bacteraemia episodes recently identified in Australia were due to LA-MRSA or CA-MRSA CC398. Isolates were sourced from the Australian Group on Antimicrobial Resistance S. aureus surveillance programme and episodes occurred across Australia. Whole-genome sequencing (WGS) and phylogenetic comparison of the ten CC398 bacteraemia isolates with previously published CC398 MRSA whole-genome sequences identified that the Australian CC398 isolates were closely related to the human-associated II-GOI clade and the livestock-associated IIa clade. The identified CC398 MRSA clones were: PVL-positive ST1232-V (5C25), PVL-negative community-associated ST398-V (5C25) and livestock-associated ST398-V (5C25). Our findings demonstrate the importance of using WGS and comparing the sequences with international sequences to distinguish between CC398 CA-MRSA and LA-MRSA and to determine the isolates' origin. Furthermore, our findings suggest that CC398 CA-MRSA has become established in the Australian community and that ST398-V (5C25) LA-MRSA is now widespread in Australian piggeries. Our study emphasises the need for national One Health antimicrobial resistance surveillance programmes to assist in monitoring the ongoing epidemiology of MRSA and other clinically significant antimicrobial-resistant organisms.

In this review we aim to summarize the current state of methods for label-free identification and functional characterization of leukocytes with biosensors and novel single cell techniques. The growing interest in this field is fueled from multiple directions, with the different aspects highlighting benefits of these novel technologies in comparison to classical methods. The advantage of label-free characterization is that labeling the cells might affect their behavior, and therefore lead to a biased description of the investigated biological phenomena. Label-free biosensors can offer the benefit of (i) decreasing processing time and reagent costs, (ii) enable point-of-care diagnostics, and (iii) allow downstream application of the investigated cells. Moreover, (iv) label-free detection allows the monitoring of real-time kinetic processes, opening up new avenues in contrast to traditional structural characterizations. The emphasis in the review will be on techniques on the characterizations of single cells with special attention to surface sensitive technologies. Recent developments highlighted the importance of small cell populations and individual cells both in health and disease. Nonetheless techniques capable of analyzing single cells offer a promising tool for therapeutic approaches where characterization of individual cells is necessary to estimate their clinical therapeutic potential. Most of the approaches discussed here will cover the cellular activation, adhesion as measured on functionalized solid substrates, since this approach offers the most advantages. Analyzing various cells on solid substrates not only allows their individual morphological characterization and therefore a more precise description of their activation, but as well offers an opportunity to design multiplex measurements. With this approach different stimuli can be investigated in parallel and measure cellular avidity to targets, an important aspect of gaining more and more attention recently in characterization of T-cells and antibody effector functions. Finally, novel label-free approaches provide a solution to extracting unlabeled cells for downstream processing (e.g., transcriptome analysis, cloning or the aforementioned clinical potential), where ongoing and potential further applications are discussed.

Accurate estimates of genome-wide rates and fitness effects of new mutations are essential for an improved understanding of molecular evolutionary processes. Although eukaryotic genomes generally contain a large noncoding fraction, functional noncoding regions and fitness effects of mutations in such regions are still incompletely characterized. A promising approach to characterize functional noncoding regions relies on identifying accessible chromatin regions (ACRs) tightly associated with regulatory DNA. Here, we applied this approach to identify and estimate selection on ACRs in Capsella grandiflora, a crucifer species ideal for population genomic quantification of selection due to its favorable population demography. We describe a population-wide ACR distribution based on ATAC-seq data for leaf samples of 16 individuals from a natural population. We use population genomic methods to estimate fitness effects and proportions of positively selected fixations (α) in ACRs and find that intergenic ACRs harbor a considerable fraction of weakly deleterious new mutations, as well as a significantly higher proportion of strongly deleterious mutations than comparable inaccessible intergenic regions. ACRs are enriched for expression quantitative trait loci (eQTL) and depleted of transposable element insertions, as expected if intergenic ACRs are under selection because they harbor regulatory regions. By integrating empirical identification of intergenic ACRs with analyses of eQTL and population genomic analyses of selection, we demonstrate that intergenic regulatory regions are an important source of nearly neutral mutations. These results improve our understanding of selection on noncoding regions and the role of nearly neutral mutations for evolutionary processes in outcrossing Brassicaceae species.

Fertilization in angiosperms involves the germination of pollen on the stigma, followed by the extrusion of a pollen tube that elongates through the style and delivers two sperm cells to the embryo sac. Sexual selection could occur throughout this process when male gametophytes compete for fertilization. The strength of sexual selection during pollen competition should be affected by the number of genotypes deposited on the stigma. As increased self-fertilization reduces the number of mating partners, and the genetic diversity and heterozygosity of populations, it should thereby reduce the intensity of sexual selection during pollen competition. Despite the prevalence of mating system shifts, few studies have directly compared the molecular signatures of sexual selection during pollen competition in populations with different mating systems. Here we analyzed whole-genome sequences from natural populations of Arabis alpina, a species showing mating system variation across its distribution, to test whether shifts from cross- to self-fertilization result in molecular signatures consistent with sexual selection on genes involved in pollen competition. We found evidence for efficient purifying selection on genes expressed in vegetative pollen, and overall weaker selection on sperm-expressed genes. This pattern was robust when controlling for gene expression level and specificity. In agreement with the expectation that sexual selection intensifies under cross-fertilization, we found that the efficacy of purifying selection on male gametophyte-expressed genes was significantly stronger in genetically more diverse and outbred populations. Our results show that intra-sexual competition shapes the evolution of pollen-expressed genes, and that its strength fades with increasing self-fertilization rates.

Characterization of the binding of functionalized microparticles to surfaces with a specific chemistry sheds light on molecular scale interactions. Polymer or protein adsorption are often monitored by colloid particle deposition. Force measurements on microbeads by atomic force microscopy (AFM) or optical tweezers are standard methods in molecular biophysics, but typically have low throughput. Washing and centrifuge assays with (bio)chemically decorated microbeads provide better statistics, but only qualitative results without a calibrated binding force or energy value. In the present work we demonstrate that a computer controlled micropipette (CCMP) is a straightforward and high-throughput alternative to quantify the surface adhesion of functionalized microparticles. However, being an indirect force measurement technique, its in-depth comparison with a direct force measurement is a prerequisite of applications requiring calibrated adhesion force values. To this end, we attached polystyrene microbeads to a solid support by the avidin-biotin linkage. We measured the adhesion strength of the microbeads with both a specialized robotic fluid force microscope (FluidFM BOT) and CCMP. Furthermore, the bead-support contact zone was directly characterized on an optical waveguide biosensor to determine the density of avidin molecules. Distribution of the detachment force recorded on ∼50 individual beads by FluidFM BOT was compared to the adhesion distribution obtained from CCMP measurements on hundreds of individual beads. We found that both methods provide unimodal histograms. We conclude that FluidFM BOT can directly measure the detachment force curve of 50 microbeads in 150 min. CCMP can provide calibrated binding/adhesion force values of 120 microbeads in an hour.

Cell-cell and cell-matrix adhesions are fundamental in all multicellular organisms. They play a key role in cellular growth, differentiation, pattern formation and migration. Cell-cell adhesion is substantial in the immune response, pathogen-host interactions, and tumor development. The success of tissue engineering and stem cell implantations strongly depends on the fine control of live cell adhesion on the surface of natural or biomimetic scaffolds. Therefore, the quantitative and precise measurement of the adhesion strength of living cells is critical, not only in basic research but in modern technologies, too. Several techniques have been developed or are under development to quantify cell adhesion. All of them have their pros and cons, which has to be carefully considered before the experiments and interpretation of the recorded data. Current review provides a guide to choose the appropriate technique to answer a specific biological question or to complete a biomedical test by measuring cell adhesion.

SALEN- and SALAN-based complexes with catalytically active metal centers are very promising small molecules to be utilized as part of antioxidant therapies. Here we discuss a modified SALAN-type molecule armed with two phosphonate groups that significantly increase its water solubility and aid to furnish mono- or dinuclear complexes with Cu2+ ions. The regulation of the SOD-mimicking (i.e., catalytic) disproportionation reaction of the superoxide radical anion (O2•−) at pH ~7.5 could be achieved by adjusting the metal-to-ligand stoichiometry as confirmed by McCord-Fridovich and pulse radiolysis tests. The higher antioxidant activity of the dicopper complex can be explained by the better access of O2•− to the copper centers and their more positive Cu(II)/Cu(I) redox potential. Simultaneously the analysis of in vitro effect on cells morphology indicates that cytotoxicity is also affected by the metal-to-ligand ratio, however, the active complex molecules do not show notable cytotoxicity that, together with the observed SOD-like activities, makes them potential candidates for antioxidant therapies.

Optical Waveguide Lightmode Spectroscopy (OWLS) is widely applied to monitor protein adsorption, polymer self-assembly, and living cells on the surface of the sensor in a label-free manner. Typically, to determine the optogeometrical parameters of the analyte layer (adlayer), the homogeneous and isotropic thin adlayer model is used to analyze the recorded OWLS data. However, in most practical situations, the analyte layer is neither homogeneous nor isotropic. Therefore, the measurement with two waveguide modes and the applied model cannot supply enough information about the parameters of the possible adlayer inhomogeneity and anisotropy. Only the so-called quasihomogeneous adlayer refractive index, layer thickness, and surface mass can be determined. In the present work, we construct an inhomogeneous adlayer model. In our model, the adlayer covers the waveguide surface only partially and it has a given refractive index profile perpendicular to the surface of the sensor. Using analytical and numerical model calculations, the step-index and exponential refractive index profiles are investigated with varying surface coverages from 0 to 100%. The relevant equations are summarized and three typically employed waveguide sensor structures are studied in detail. We predict the errors in the calculated optogeometrical parameters of the adlayer by simulating the OWLS measurement on an assumed inhomogeneous adlayer. We found that the surface coverage has negligible influence on the calculated refractive index below film thicknesses of 5 nm; the calculated refractive index is close to the refractive index of the adlayer islands. But the determined quasihomogeneous adlayer refractive index and surface mass are always underrated; the calculated quasihomogeneous thickness is heavily influenced by the surface coverage. Depending on the refractive index profile, waveguide geometry, and surface coverage, the thickness obtained from the homogeneous and isotropic modeling can even take negative and largely overestimated values, too. Therefore, experimentally obtained unrealistic adlayer values, which were dismissed previously, might be important indicators of layer structure.

Micropatterning of living single cells and cell clusters over millimeter-centimeter scale areas is of high demand in the development of cell-based biosensors. Micropatterning methodologies require both a suitable biomimetic support and a printing technology. In this work, we present the micropatterning of living mammalian cells on carboxymethyl dextran (CMD) hydrogel layers using the FluidFM BOT technology. In contrast to the ultrathin (few nanometers thick in the dry state) CMD films generally used in label-free biosensor applications, we developed CMD layers with thicknesses of several tens of nanometers in order to provide support for the controlled adhesion of living cells. The fabrication method and detailed characterization of the CMD layers are also described. The antifouling ability of the CMD surfaces is demonstrated by in situ optical waveguide lightmode spectroscopy measurements using serum modeling proteins with different electrostatic properties and molecular weights. Cell micropatterning on the CMD surface was obtained by printing cell adhesion mediating cRGDfK peptide molecules (cyclo(Arg-Gly-Asp-d-Phe-Lys)) directly from aqueous solution using microchanneled cantilevers with subsequent incubation of the printed surfaces in the living cell culture. Uniquely, we present cell patterns with different geometries (spot, line, and grid arrays) covering both micrometer and millimeter-centimeter scale areas. The adhered patterns were analyzed by phase contrast microscopy and the adhesion process on the patterns was real-time monitored by digital holographic microscopy, enabling to quantify the survival and migration of cells on the printed cRGDfK arrays.

The epithelium covers, protects, and actively regulates various formations and cavities of the human body. During embryonic development the assembly of the epithelium is crucial to the organoid formation, and the invasion of the epithelium is an essential step in cancer metastasis. Live cell mechanical properties and associated forces presumably play an important role in these biological processes. However, the direct measurement of cellular forces in a precise and high-throughput manner is still challenging. We studied the cellular adhesion maturation of epithelial Vero monolayers by measuring single-cell force-spectra with high-throughput fluidic force microscopy (robotic FluidFM). Vero cells were grown on gelatin-covered plates in different seeding concentrations, and cell detachment forces were recorded from the single-cell state, through clustered island formation, to their complete assembly into a sparse and then into a tight monolayer. A methodology was proposed to separate cell-substratum and cell-cell adhesion force and energy (work of adhesion) contributions based on the recorded force-distance curves. For comparison, cancerous HeLa cells were also measured in the same settings. During Vero monolayer formation, a significantly strengthening adhesive tendency was found, showing the development of cell-cell contacts. Interestingly, this type of step-by-step maturation was absent in HeLa cells. The attachment of cancerous HeLa cells to the assembled epithelial monolayers was also measured, proposing a new high-throughput method to investigate the biomechanics of cancer cell invasion. We found that HeLa cells adhere significantly stronger to the tight Vero monolayer than cells of the same origin. Moreover, the mechanical characteristics of Vero monolayers upon cancerous HeLa cell influence were recorded and analyzed. All these results provide insight into the qualitative assessment of cell-substratum and cell-cell mechanical contacts in mono- and multilayered assemblies and demonstrate the robustness and speed of the robotic FluidFM technology to reveal biomechanical properties of live cell assemblies with statistical significances.

Numerical simulations and analytical calculations are performed to support the design of grating-coupled planar optical waveguides for biological sensing. Near cut-off and far from cut-off modes are investigated, and their characteristics and suitability for sensing are compared. The numerical simulations reveal the high sensitivity of the guided mode intensity near the cut-off wavelength for any refractive index change along the waveguide. Consequently, it is sufficient to monitor the intensity change of the near cut-off sensing mode, which leads to a simpler sensor design compared to those setups where the resonant wavelength shift of the guided mode is monitored with high precision. The operating wavelength and the sensitivity of the proposed device can be tuned by varying the geometrical parameters of the corrugated waveguide. These results may lead to the development of highly sensitive integrated sensors, which have a simple design and therefore are cost-effective for a wide range of applications. These numerical findings are supported with experimental results, where the cut-off sensing mode was identified.

A piezoelectric electro-optomechanical crystal made of Indium Gallium Phoshpide is demonstrated. The electromechanical actuation results in a coupling rate equal to 1µHz and is used for injection locking by an external generator. © 2021 The Author(s)

The robotized version of the nanofluidic force-microscope, FluidFM BOT, is a versatile tool utilized for single-cell force-spectroscopy, micropatterning, and fluid injection or extraction. It can manipulate fluids with an embedded nano-channel in the core of its special silicon-nitride cantilever by having a fluid reservoir connected to a pressure control system. The precise determination of the spring constant is essential for any cantilever. The aim of our current work is to lay the bases of a spring constant calibration method that requires knowing the first two resonance frequencies of the cantilever in air and in a viscous medium. For this purpose, we used finite element method (FEM) numerical simulation on a CAD model, which was created based on SEM images made on the FluidFM micropipette cantilever. The resonance frequencies of the cantilever in air and in a viscous medium were calculated from the simulations. The resonance frequencies of the cantilever were determined in air and water, with respect to changes in the mesh size, Young’s modulus of the cantilever, the aperture size, and the volume of surrounding media. The relevance of the data is discussed in light of experimentally determined resonance frequencies and technological limitations.

Today, there is an intense demand for lab-on-a-chip and tissue-on-a-chip applications in basic cell biological research and medical diagnostics. A particular challenge is the implementation of advanced biosensor techniques in point-of-care testing utilizing human primary cells. In this study, a resonant waveguide grating (RWG)-based label-free optical biosensor technique has been applied for real-time monitoring of the integrated responses of primary human tonsillar B cells initiated by B cell receptor (BCR) and modified by FcγRIIb and CR1 engagement. The BCR-triggered biosensor responses of resting and activated B cells were revealed to be specific and dose-dependent, in some cases with strong donor dependency. Targeted inhibition of Syk attenuated the label-free biosensor response upon BCR stimulation. Indifferent protein human serum albumin (HSA) did not interfere with the recorded signal to BCR stimulation. Simultaneous engagement of BCR and FcγRIIb modulated the kinetic signal of the cells. Activated and resting B cells exhibited different response profiles upon simultaneous engagement of BCR and CR1. This advanced approach has the potential to decipher interfering signaling events in human B cells, manage differences between activated and resting B cell states, helping to understand the actual integrated response of these immune cells, and could be useful in the point-of-care diagnostic testing on human primary cells.

Binding force between biomolecules has a crucial role in most biological processes. Receptor-ligand interactions transmit physical forces and signals simultaneously. Previously, we employed a robotic micropipette both in live cell and microbead adhesion studies to explore the adhesion force of biomolecules such as cell surface receptors including specific integrins on immune cells. Here we apply standard computational fluid dynamics simulations to reveal the detailed physical background of the flow generated by the micropipette when probing microbead adhesion on functionalized surfaces. Measuring the aspiration pressure needed to pick up the biotinylated 10 μm beads on avidin coated surfaces and converting it to a hydrodynamic lifting force on the basis of simulations, we found an unbinding force of 12 ± 2 nN, when targeting the beads manually; robotic targeting resulted in 9 ± 4 nN (mean ± SD). We measured and simulated the effect of the targeting offset, when the microbead was out of the axis (off-axis)of the micropipette. According to the simulations, the higher offset resulted in a higher lifting force acting on the bead. Considering this effect, we could readily correct the impact of the targeting offset to renormalize the experimental data. Horizontal force and torque also appeared in simulations in case of a targeting offset. Surprisingly, simulations show that the lifting force acting on the bead reaches a maximum at a flow rate of ~ 5 μl/s if the targeting offset is not very high (

The binding of integrin proteins to peptide sequences such as arginine-glycine-aspartic acid (RGD) is a crucial step in the adhesion process of mammalian cells. While these bonds can be examined between purified proteins and their ligands, live-cell assays are better suited to gain biologically relevant information. Here we apply a computer-controlled micropipette (CCMP) to measure the dissociation constant (Kd) of integrin-RGD-binding. Surface coatings with varying RGD densities were prepared, and the detachment of single cells from these surfaces was measured by applying a local flow inducing hydrodynamic lifting force on the targeted cells in discrete steps. The average behavior of the populations was then fit according to the chemical law of mass action. To verify the resulting value of Kd2d = (4503 &plusmn, 1673) 1/&micro, m2, a resonant waveguide grating based biosensor was used, characterizing and fitting the adhesion kinetics of the cell populations. Both methods yielded a Kd within the same range. Furthermore, an analysis of subpopulations was presented, confirming the ability of CCMP to characterize cell adhesion both on single cell and whole population levels. The introduced methodologies offer convenient and automated routes to quantify the adhesivity of living cells before their further processing.

The glycocalyx is thought to perform a potent, but not yet defined function in cellular adhesion and signaling. Since 95% of cancer cells have altered glycocalyx structure, this role can be especially important in cancer development and metastasis. The glycocalyx layer of cancer cells directly influences cancer progression, involving the complicated kinetic process of cellular adhesion at various levels. In the present work, we investigated the effect of enzymatic digestion of specific glycocalyx components on cancer cell adhesion to RGD (arginine–glycine–aspartic acid) peptide motif displaying surfaces. High resolution kinetic data of cell adhesion was recorded by the surface sensitive label-free resonant waveguide grating (RWG) biosensor, supported by fluorescent staining of the cells and cell surface charge measurements. We found that intense removal of chondroitin sulfate (CS) and dermatan sulfate chains by chondroitinase ABC reduced the speed and decreased the strength of adhesion of HeLa cells. In contrast, mild digestion of glycocalyx resulted in faster and stronger adhesion. Control experiments on a healthy and another cancer cell line were also conducted, and the discrepancies were analysed. We developed a biophysical model which was fitted to the kinetic data of HeLa cells. Our analysis suggests that the rate of integrin receptor transport to the adhesion zone and integrin-RGD binding is strongly influenced by the presence of glycocalyx components, but the integrin-RGD dissociation is not. Moreover, based on the kinetic data we calculated the dependence of the dissociation constant of integrin-RGD binding on the enzyme concentration. We also determined the dissociation constant using a 2D receptor binding model based on saturation level static data recorded at surfaces with tuned RGD densities. We analyzed the discrepancies of the kinetic and static dissociation constants, further illuminating the role of cancer cell glycocalyx during the adhesion process. Altogether, our experimental results and modelling demonstrated that the chondroitin sulfate and dermatan sulfate chains of glycocalyx have an important regulatory function during the cellular adhesion process, mainly controlling the kinetics of integrin transport and integrin assembly into mature adhesion sites. Our results potentially open the way for novel type of cancer treatments affecting these regulatory mechanisms of cellular glycocalyx.

The robotized fluidic-force microscopy (FluidFM BOT) technology enables label-free, high-throughput, and flexible data acquisition, and have found a wide range of applications in single-cell biology. One of its features is to measure the characteristic force-distance (FD) curves produced by living cells during single-cell force-spectroscopy (SCFS). In the present work, African green monkey kidney epithelial (Vero) cells were seeded on a gelatin-coated substrate to mimic their attachment to the extracellular matrix. Vero cells were detached from the substrate with 8 μm aperture micropipette cantilever, and their FD-curves were investigated besides the actual cell size. The results on 19 individual cells indicate that cell size and adhesion force may deviate; however, highly adhered cells require a greater distance to detach them compared to weakly adhered cells. Adhesion force and energy of the cell normalized by their cell area also show rising linear tendencies with the detachment distance. The investigation presents how cellular morphology influences the adhesive properties of Vero cells.

Background The optimal antibiotic treatment length for infective endocarditis (IE) is uncertain. International guidelines recommend treatment duration of up to 6 weeks for patients with left-sided IE but are primarily based on historical data and expert opinion. Efficacies of modern therapies, fast recovery seen in many patients with IE, and complications to long hospital stays challenge the rationale for fixed treatment durations in all patients. Objective The objective was to conduct a noninferiority randomized controlled trial (acronym POET II) investigating the safety of accelerated (shortened) antibiotic therapy as compared to standard duration in patients with left-sided IE. Methods The POET II trial is a multicenter, multinational, open-label, noninferiority randomized controlled trial. Patients with definite left-sided IE due to Streptococcus spp, Staphylococcus aureus, or Enterococcus faecalis will be eligible for enrolment. Each patient will be randomized to accelerated antibiotic treatment or standard-length treatment (1:1) following clinical stabilization as defined by clinical parameters, laboratory values, and transesophageal echocardiography findings. Accelerated treatment will be between 2 and 4 weeks, whereas standard-length treatment will be between 4 and 6 weeks, depending on microbiologic etiology, complications, need for valve surgery, and prosthetic versus native valve endocarditis. The primary outcome is a composite of all-cause mortality, unplanned cardiac surgery, relapse of bacteremia, or embolization within 6 months of randomization. Conclusions The POET II trial will investigate the safety of accelerated antibiotic therapy for patients with left-sided IE caused by Streptococcus spp, Staphylococcus aureus, or Enterococcus faecalis. The results of the POET II trial will improve the evidence base of treatment recommendations, and clinical practice may be altered.

Cellular monolayers have a fundamental role in the development of embryos, vascularization, organ formation, attachment to artificial implants, and metastasis of cancerous cells. Phase holographic imaging was used to monitor the time-dependent behavior of cancerous HeLa cells. After monitoring the assembly of epithelial Vero monolayer on a gelatin-coated surface, HeLa cells were seeded on top of the monolayer, and their transmigration was observed. This method is label-free and non-toxic to cells and enables the visualization of living cells in real-time and analyze their parameters such as motility and morphology. HeLa cells seeded on the tight Vero monolayer (100 % confluency) were observed for 24 hours, and a 60 minute time period has been selected for further analysis that showed the difference in cellular parameters between non-invasive and invasive HeLa cells in the observed time frame. Our investigations revealed that invasive HeLa cells have reduced area and optical volume compared to noninvasive HeLa cells, corresponding to the phase shift detected in transmigration events.

Although microliter-scale liquid handling with a handheld pipette is a routine task, pipetting nanoliter-scale volumes is challenging due to several technical difficulties including surface tension, adhesion and evaporation effects. We developed a fully automated piezoelectric micropipette with a precision of

Biofunctional coatings are key elements of biosensors regulating interactions between the sensing surface and analytes as well as matrix components of the sample. These coatings can improve sensing capabilities both by amplifying the target signal and attenuating interfering signals originating from surface fouling (non-specific binding). Considering the tested materials so far, hydrogel-based layers have been verified to be among the most effective layers in improving biochip performance. The polysaccharide dextran can be efficiently used to form hydrogel layers displaying extended three-dimensional structure on biosensor surfaces. Owing to their high water content and flexible structure, dextran coatings present advanced antifouling abilities, which can be exploited in classic bioanalytical measurements as well as in the development of cell-on-a-chip type biosensors. However, in spite of the numerous applications, the deep characterization of dextran layers has been missing from the literature. This phenomenon can be attributed to the challenging analysis of few nanometer-thick layers with high water content. The lack of available data is more pronounced regarding the layer behaviors under aqueous conditions. In this chapter we present various surface analytical methods (including biosensor-type techniques) suitable for the complex characterization of hydrogel coatings whose thickness ranges from few to several ten nanometers. As a case study, we focus on the analysis of carboxymethyl dextran (CMD) layers developed for waveguide-based label-free optical biosensor applications. Examination methodologies both under dry and aqueous conditions as well as testing of antifouling abilities are also presented.