Continuous distribution of cancer cells in the cell cycle unveiled by AI-segmented imaging of 37,000 HeLa FUCCI cells.

Classification of live or fixed cells based on their unlabeled microscopic images would be a powerful tool for cell biology and pathology. For such software, the first step is the generation of a ground truth database that can be used for training and testing AI classification algorithms. The Application of cells expressing fluorescent reporter proteins allows the building of ground truth datasets in a straightforward way. In this study, we present an automated imaging pipeline utilizing the Cellpose algorithm for the precise cell segmentation and measurement of fluorescent cellular intensities across multiple channels. We analyzed the cell cycle of HeLa-FUCCI cells expressing fluorescent red and green reporter proteins at various levels depending on the cell cycle state. To build the dataset, 37,000 fixed cells were automatically scanned using a standard motorized microscope, capturing phase contrast and fluorescent red/green images. The fluorescent pixel intensity of each cell was integrated to calculate the total fluorescence of cells based on cell segmentation in the phase contrast channel. It resulted in a precise intensity value for each cell in both channels. Furthermore, we conducted a comparative analysis of Cellpose 1.0 and Cellpose 2.0 in cell segmentation performance. Cellpose 2.0 demonstrated notable improvements, achieving a significantly reduced false positive rate of 2.7 % and 1.4 % false negative. The cellular fluorescence was visualized in a 2D plot (map) based on the red and green intensities of the FUCCI construct revealing the continuous distribution of cells in the cell cycle. This 2D map enables the selection and potential isolation of single cells in a specific phase. In the corresponding heatmap, two clusters appeared representing cells in the red and green states. Our pipeline allows the high-throughput and accurate measurement of cellular fluorescence providing extensive statistical information on thousands of cells with potential applications in developmental and cancer biology. Furthermore, our method can be used to build ground truth datasets automatically for training and testing AI cell classification. Our automated pipeline can be used to analyze thousands of cells within 2 h after putting the sample onto the microscope.

Optical sensor reveals the hidden influence of cell dissociation on adhesion measurements.

Cell adhesion experiments are important in tissue engineering and for testing new biologically active surfaces, prostheses, and medical devices. Additionally, the initial state of adhesion (referred to as nascent adhesion) plays a key role and is currently being intensively researched. A critical step in handling all adherent cell types is their dissociation from their substrates for further processing. Various cell dissociation methods and reagents are used in most tissue culture laboratories (here, cell dissociation from the culture surface, cell harvesting, and cell detachment are used interchangeably). Typically, the dissociated cells are re-adhered for specific measurements or applications. However, the impact of the choice of dissociation method on cell adhesion in subsequent measurements, especially when comparing the adhesivity of various surfaces, is not well clarified. In this study, we demonstrate that the application of a label-free optical sensor can precisely quantify the effect of cell dissociation methods on cell adhesivity, both at the single-cell and population levels. The optical measurements allow for high-resolution monitoring of cellular adhesion without interfering with the physiological state of the cells. We found that the choice of reagent significantly alters cell adhesion on various surfaces. Our results clearly demonstrate that biological conclusions about cellular adhesion when comparing various surfaces are highly dependent on the employed dissociation method. Neglecting the choice of cellular dissociation can lead to misleading conclusions when evaluating cell adhesion data from various sources and comparing the adhesivity of two different surfaces (i.e., determining which surface is more or less adhesive).

Single-cell classification based on label-free high-resolution optical data of cell adhesion kinetics.

Selecting and isolating various cell types is a critical procedure in many applications, including immune therapy, regenerative medicine, and cancer research. Usually, these selection processes involve some labeling or another invasive step potentially affecting cellular functionality or damaging the cell. In the current proof of principle study, we first introduce an optical biosensor-based method capable of classification between healthy and numerous cancerous cell types in a label-free setup. We present high classification accuracy based on the monitored single-cell adhesion kinetic signals. We developed a high-throughput data processing pipeline to build a benchmark database of ~ 4500 single-cell adhesion measurements of a normal preosteoblast (MC3T3-E1) and various cancer (HeLa, LCLC-103H, MDA-MB-231, MCF-7) cell types. Several datasets were used with different cell-type selections to test the performance of deep learning-based classification models, reaching above 70-80% depending on the classification task. Beyond testing these models, we aimed to draw interpretable biological insights from their results; thus, we applied a deep neural network visualization method (grad-CAM) to reveal the basis on which these complex models made their decisions. Our proof-of-concept work demonstrated the success of a deep neural network using merely label-free adhesion kinetic data to classify single mammalian cells into different cell types. We propose our method for label-free single-cell profiling and in vitro cancer research involving adhesion. The employed label-free measurement is noninvasive and does not affect cellular functionality. Therefore, it could also be adapted for applications where the selected cells need further processing, such as immune therapy and regenerative medicine.

Label-free biomolecular and cellular methods in small molecule epigallocatechin-gallate research.

Small molecule natural compounds are gaining popularity in biomedicine due to their easy access to wide structural diversity and their proven health benefits in several case studies. Affinity measurements of small molecules below 100 Da molecular weight in a label-free and automatized manner using small amounts of samples have now become a possibility and reviewed in the present work. We also highlight novel label-free setups with excellent time resolution, which is important for kinetic measurements of biomolecules and living cells. We summarize how molecular-scale affinity data can be obtained from the in-depth analysis of cellular kinetic signals. Unlike traditional measurements, label-free biosensors have made such measurements possible, even without the isolation of specific cellular receptors of interest. Throughout this review, we consider epigallocatechin gallate (EGCG) as an exemplary compound. EGCG, a catechin found in green tea, is a well-established anti-inflammatory and anti-cancer agent. It has undergone extensive examination in numerous studies, which typically rely on fluorescent-based methods to explore its effects on both healthy and tumor cells. The summarized research topics range from molecular interactions with proteins and biological films to the kinetics of cellular adhesion and movement on novel biomimetic interfaces in the presence of EGCG. While the direct impact of small molecules on living cells and biomolecules is relatively well investigated in the literature using traditional biological measurements, this review also highlights the indirect influence of these molecules on the cells by modifying their nano-environment. Moreover, we underscore the significance of novel high-throughput label-free techniques in small molecular measurements, facilitating the investigation of both molecular-scale interactions and cellular processes in one single experiment. This advancement opens the door to exploring more complex multicomponent models that were previously beyond the reach of traditional assays.

Nanoinjection of extracellular vesicles to single live cells by robotic fluidic force microscopy.

In the past decade, extracellular vesicles (EVs) have attracted substantial interest in biomedicine. With progress in the field, we have an increasing understanding of cellular responses to EVs. In this Technical Report, we describe the direct nanoinjection of EVs into the cytoplasm of single cells of different cell lines. By using robotic fluidic force microscopy (robotic FluidFM), nanoinjection of GFP positive EVs and EV-like particles into single live HeLa, H9c2, MDA-MB-231 and LCLC-103H cells proved to be feasible. This injection platform offered the advantage of high cell selectivity and efficiency. The nanoinjected EVs were initially localized in concentrated spot-like regions within the cytoplasm. Later, they were transported towards the periphery of the cells. Based on our proof-of-principle data, robotic FluidFM is suitable for targeting single living cells by EVs and may lead to information about intracellular EV cargo delivery at a single-cell level.

Polysaccharide-based nano-engineered multilayers for controlled cellular adhesion in label-free biosensors.

Controlling cellular adhesion is a critical step in the development of biomaterials, and in cell- based biosensing assays. Usually, the adhesivity of cells is tuned by an appropriate biocompatible layer. Here, synthetic poly(diallyldimethylammonium chloride) (PDADMAC), natural chitosan, and heparin (existing in an extracellular matrix) were selected to assembly PDADMAC/heparin and chitosan/heparin films. The physicochemical properties of macroion multilayers were determined by streaming potential measurements (SPM), quartz crystal microbalance (QCM-D), and optical waveguide lightmode spectroscopy (OWLS). The topography of the wet films was imaged using atomic force microscopy (AFM). The adhesion of preosteoblastic cell line MC3T3-E1 on those well-characterized polysaccharide-based multilayers was evaluated using a resonant waveguide grating (RWG) based optical biosensor and digital holographic microscopy. The latter method was engaged to investigate long-term cellular behavior on the fabricated multilayers. (PDADMAC/heparin) films were proved to be the most effective in inducing cellular adhesion. The cell attachment to chitosan/heparin-based multilayers was negligible. It was found that efficient adhesion of the cells occurs onto homogeneous and rigid multilayers (PDADMAC/heparin), whereas the macroion films forming "sponge-like" structures (chitosan/heparin) are less effective, and could be employed when reduced adhesion is needed. Polysaccharide-based multilayers can be considered versatile systems for medical applications. One can postulate that the presented results are relevant not only for modeling studies but also for applied research.

Label-free biosensing of lignans for therapeutics using engineered model surfaces.

The label-free interaction analysis of macromolecules and small molecules has increasing importance nowadays, both in diagnostics and therapeutics. In the blood vascular system, human serum albumin (HSA) is a vital globular transport protein with potential multiple ligand binding sites. Characterizing the binding affinity of compounds to HSA is essential in pharmaceutics and in developing new compounds for clinical application. Aryltetralin lignans from the roots of Anthriscus sylvestris are potential antitumor therapeutic candidates, but their molecular scale interactions with specific biomolecules are unrevealed. Here, we applied the label-free grating-coupled interferometry (GCI) biosensing method with a polycarboxylate-based hydrogel layer with immobilized HSA on top of it. With this engineered model surface, we could determine the binding parameters of two novel aryltetralin lignans, deoxypodophyllotoxin (DPT), and angeloyl podophyllotoxin (APT) to HSA. Exploiting the multi-channel referencing ability, the unique surface sensitivity, and the throughput of GCI, we first revealed the specific biomolecular interactions. Traditional label-free kinetic measurements were also compared with a novel, fast way of measuring affinity kinetics using less sample material (repeated analyte pulses of increasing duration (RAPID)). Experiments with well-characterized molecular interactions (furosemide to carbonic-anhydrase (CAII) and warfarin, norfloxacin to HSA) were performed to prove the reliability of the RAPID method. In all investigated cases, the RAPID and traditional measurement gave similar affinity values. In the case of DPT, the measurements and relevant modeling suggested two binding sites on HSA, with dissociation constant values of Kd1 = 1.8 ± 0.01 µM, Kd2 = 3 ± 0.02 µM. In the case of APT, the experiments resulted in Kd1 = 9 ± 1.7 µM, Kd2 = 28 ± 0.3 µM. The obtained binding values might suggest the potential medical application of DPT and APT without further optimization of their binding affinity to HSA. These results could be also adapted to other biomolecules and applications where sample consumption and the rapidity of the measurements are critical.

Dean-Flow Affected Lateral Focusing and Separation of Particles and Cells in Periodically Inhomogeneous Microfluidic Channels.

The purpose of the recent work is to give a better explanation of how Dean vortices affect lateral focusing, and to understand how cell morphology can alter the focusing position compared to spherical particles. The position and extent of the focused region were investigated using polystyrene fluorescent beads with different bead diameters (Ø = 0.5, 1.1, 1.97, 2.9, 4.8, 5.4, 6.08, 10.2, 15.8, 16.5 µm) at different flow rates (0.5, 1, 2 µL/s). Size-dependent focusing generated a precise map of the equilibrium positions of the spherical beads at the end of the periodically altering channels, which gave a good benchmark for focusing multi-dimensional particles and cells. The biological samples used for experiments were rod-shaped Escherichia coli (E. coli), discoid biconcave-shaped red blood cells (RBC), round or ovoid-shaped yeast, Saccharomyces cerevisiae, and soft-irregular-shaped HeLa cancer-cell-line cells to understand how the shape of the cells affects the focusing position at the end of the channel.

Glycocalyx Components Detune the Cellular Uptake of Gold Nanoparticles in a Size- and Charge-Dependent Manner.

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 [d = 2.6, 4.2, and 7.0 nm, small (S), medium (M), and large(L), respectively]. Negatively charged citrate-capped tannic acid (CTA, d = 5.5 nm) NPs were also used in control experiments. Real-time biosensor data confirmed the cellular uptake of the functionalized NPs, which was visually proved by transmission electron microscopy. It was found that the enzymatic digestion facilitated the entry of the positively charged S- and M-sized NPs, being more pronounced for the M-sized. Other enzymes digesting different components of the glycocalyx were also employed, and the results were compared. Glycosaminoglycan digesting heparinase III treatment also increased, while glycoprotein and glycolipid modifying neuraminidase decreased the NP uptake by HeLa cells. This suggests that the sialic acid residues increase, while heparan sulfate decreases the uptake of positively charged NPs. Our results raise the hypothesis that cellular uptake of 2-4 nm positively charged NPs is facilitated by glycoprotein and glycolipid components of the glycocalyx but inhibited by PGs.

Comparative Assessment of the Inhibitory Potential of the Herbicide Glyphosate and Its Structural Analogs on RGD-Specific Integrins Using Enzyme-Linked Immunosorbent Assays.

Transmembrane glycoprotein integrins play crucial roles in biochemical processes, and by their inhibition or activation, different signal pathways can be disrupted, leading to abnormal physiological functions. We have previously demonstrated the inhibitory effect of glyphosate herbicide's active ingredient on cell adhesion and its αvß3 integrin antagonist effect. Therefore, it appeared particularly exciting to investigate inhibition of glyphosate and its metabolites on a wider range of Arg-Gly-Asp (RGD) binding integrins, namely αvß3, α5ß1 and αllbß3. Thus, the purpose of this study was to assess how extended the inhibitory effect observed for glyphosate on the integrin αvß3 is in terms of other RGD integrins and other structurally or metabolically related derivatives of glyphosate. Five different experimental setups using enzyme-linked immunosorbent assays were applied: (i) αvß3 binding to a synthetic polymer containing RGD; (ii) αvß3 binding to its extracellular matrix (ECM) protein, vitronectin; (iii) α5ß1 binding to the above polymer containing RGD; (iv) αllbß3 binding to its ECM protein, fibrinogen and (v) αvß3 binding to the SARS-CoV-2 spike protein receptor binding domain. Total inhibition of αvß3 binding to RGD was detected for glyphosate and its main metabolite, aminomethylphosphonic acid (AMPA), as well as for acetylglycine on α5ß1 binding to RGD.

Cell-substratum and cell-cell adhesion forces and single-cell mechanical properties in mono- and multilayer assemblies from robotic fluidic force microscopy.

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.

Functional blood cell analysis by label-free biosensors and single-cell technologies.

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.

Cytotoxic effects of Roundup Classic and its components on NE-4C and MC3T3-E1 cell lines determined by biochemical and flow cytometric assays.

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.

Simple and automatic monitoring of cancer cell invasion into an epithelial monolayer using label-free holographic microscopy.

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.

Population distributions of single-cell adhesion parameters during the cell cycle from high-throughput robotic fluidic force microscopy.

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.

Review of Label-Free Monitoring of Bacteria: From Challenging Practical Applications to Basic Research Perspectives.

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.

Development and In-Depth Characterization of Bacteria Repellent and Bacteria Adhesive Antibody-Coated Surfaces Using Optical Waveguide Biosensing.

Bacteria repellent surfaces and antibody-based coatings for bacterial assays have shown a growing demand in the field of biosensors, and have crucial importance in the design of biomedical devices. However, in-depth investigations and comparisons of possible solutions are still missing. The optical waveguide lightmode spectroscopy (OWLS) technique offers label-free, non-invasive, in situ characterization of protein and bacterial adsorption. Moreover, it has excellent flexibility for testing various surface coatings. Here, we describe an OWLS-based method supporting the development of bacteria repellent surfaces and characterize the layer structures and affinities of different antibody-based coatings for bacterial assays. In order to test nonspecific binding blocking agents against bacteria, OWLS chips were coated with bovine serum albumin (BSA), I-block, PAcrAM-g-(PMOXA, NH2, Si), (PAcrAM-P) and PLL-g-PEG (PP) (with different coating temperatures), and subsequent Escherichia coli adhesion was monitored. We found that the best performing blocking agents could inhibit bacterial adhesion from samples with bacteria concentrations of up to 107 cells/mL. Various immobilization methods were applied to graft a wide range of selected antibodies onto the biosensor's surface. Simple physisorption, Mix&Go (AnteoBind) (MG) films, covalently immobilized protein A and avidin-biotin based surface chemistries were all fabricated and tested. The surface adsorbed mass densities of deposited antibodies were determined, and the biosensor;s kinetic data were evaluated to divine the possible orientations of the bacteria-capturing antibodies and determine the rate constants and footprints of the binding events. The development of affinity layers was supported by enzyme-linked immunosorbent assay (ELISA) measurements in order to test the bacteria binding capabilities of the antibodies. The best performance in the biosensor measurements was achieved by employing a polyclonal antibody in combination with protein A-based immobilization and PAcrAM-P blocking of nonspecific binding. Using this setting, a surface sensitivity of 70 cells/mm2 was demonstrated.

Single-cell adhesion strength and contact density drops in the M phase of cancer cells.

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.

Label-free real-time monitoring of the BCR-triggered activation of primary human B cells modulated by the simultaneous engagement of inhibitory receptors.

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.

Data evaluation for surface-sensitive label-free methods to obtain real-time kinetic and structural information of thin films: A practical review with related software packages.

Interfacial layers are important in a wide range of applications in biomedicine, biosensing, analytical chemistry and the maritime industries. Given the growing number of applications, analysis of such layers and understanding their behavior is becoming crucial. Label-free surface sensitive methods are excellent for monitoring the formation kinetics, structure and its evolution of thin layers, even at the nanoscale. In this paper, we review existing and commercially available label-free techniques and demonstrate how the experimentally obtained data can be utilized to extract kinetic and structural information during and after formation, and any subsequent adsorption/desorption processes. We outline techniques, some traditional and some novel, based on the principles of optical and mechanical transduction. Our special focus is the current possibilities of combining label-free methods, which is a powerful approach to extend the range of detected and deduced parameters. We summarize the most important theoretical considerations for obtaining reliable information from measurements taking place in liquid environments and, hence, with layers in a hydrated state. A thorough treamtmaent of the various kinetic and structural quantities obtained from evaluation of the raw label-free data are provided. Such quantities include layer thickness, refractive index, optical anisotropy (and molecular orientation derived therefrom), degree of hydration, viscoelasticity, as well as association and dissociation rate constants and occupied area of subsequently adsorbed species. To demonstrate the effect of variations in model conditions on the observed data, simulations of kinetic curves at various model settings are also included. Based on our own extensive experience with optical waveguide lightmode spectroscopy (OWLS) and the quartz crystal microbalance (QCM), we have developed dedicated software packages for data analysis, which are made available to the scientific community alongside this paper.