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Abstract 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).

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.

Abstract 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.

Abstract 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.

Abstract 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.

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.

Abstract 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 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.

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.

The field of extracellular vesicles (EVs) is an exponentially growing segment of biomedical sciences. However, the problems of normalisation and quantification of EV samples have not been completely solved. Currently, EV samples are standardised on the basis of their protein content sometimes combined with determination of the particle number. However, even this combined approach may result in inaccuracy and overestimation of the EV concentration. Lipid bilayers are indispensable components of EVs. Therefore, a lipid-based quantification, in combination with the determination of particle count and/or protein content, appears to be a straightforward and logical approach for the EV field. In this study, we set the goal to improve the previously reported sulfo-phospho-vanillin (SPV) lipid assay. We introduced an aqueous phase liposome standard (DOPC) to replace the purified lipid standards in organic solvents (used commonly in previous studies). Furthermore, we optimised the concentration of the vanillin reagent in the assay. We found that elimination of organic solvents from the reaction mixture could abolish the background colour that interfered with the assay. Comparison of the optimised assay with a commercial lipid kit (based on the original SPV lipid assay) showed an increase of sensitivity by approximately one order of magnitude. Thus, here we report a quick, reliable and sensitive test that may fill an existing gap in EV standardisation. When using the optimised lipid assay reported here, EV lipid measurements can be more reliable than protein-based measurements. Furthermore, this novel assay is almost as sensitive and as easy as measuring proteins with a simple BCA test.

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 ± 1673) 1/µm2, 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.

Novel optical waveguide lightmode spectroscopy (OWLS)-based immunosensor formats were developed for label-free detection of Fusarium mycotoxin zearalenone (ZON). To achieve low limits of detection (LODs), both immobilised antibody-based (direct) and immobilised antigen-based (competitive) assay setups were applied. Immunoreagents were immobilised on epoxy-, amino-, and carboxyl-functionalised sensor surfaces, and by optimising the immobilisation methods, standard sigmoid curves were obtained in both sensor formats. An outstanding LOD of 0.002 pg/mL was obtained for ZON in the competitive immunosensor setup with a dynamic detection range between 0.01 and 1 pg/mL ZON concentrations, depending on the covalent immobilisation method applied. This corresponds to a five orders of magnitude improvement in detectability of ZON relative to the previously developed enzyme-linked immonosorbent assay (ELISA) method. The selectivity of the immunosensor for ZON was demonstrated with structural analogues (α-zearalenol, α-zearalanol, and β-zearalanol) and structurally unrelated mycotoxins. The method was found to be applicable in maize extract using acetonitrile as the organic solvent, upon a dilution rate of 1:10,000 in buffer. Thus, the OWLS immunosensor method developed appears to be suitable for the quantitative determination of ZON in aqueous medium. The new technique can widen the range of sensoric detection methods of ZON for surveys in food and environmental safety assessment.

Optical waveguide lightmode spectroscopic (OWLS) techniques were probed for monitoring ion permeation through channels incorporated into artificial lipid environment. A novel sensor set-up was developed by depositing liposomes or cell-derived membrane fragments onto hydrophilic polytetrafluoroethylene (PTFE) membrane. The fibrous material of PTFE membrane could entrap lipoid vesicles and the water-filled pores provided environment for the hydrophilic domains of lipid-embedded proteins. The sensor surface was kept clean from the lipid holder PTFE membrane by a water- and ion-permeable polyethylene terephthalate (PET) mesh. The sensor set-up was tested with egg yolk lecithin liposomes containing gramicidin ion channels and with cell-derived membrane fragments enriched in GABA-gated anion channels. The method allowed monitoring the move of Na(+) and organic cations through gramicidin channels and detecting the Cl(-)-channel functions of the (α5β2γ2) GABAA receptor in the presence or absence of GABA and the competitive GABA-blocker bicuculline.

Abstract 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.

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.

Abstract 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 (K d), tetrameric FliC-NikRC molecules possess high-affinity binding sites with K d 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.

Abstract 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.

Abstract 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.

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.

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.