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.

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.

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.

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.

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.

Human primary endothelial label-free biochip assay reveals unpredicted functions of plasma serine proteases.

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 kinetics of cell populations from combined label-free optical biosensor and robotic fluidic force microscopy.

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.

Oxidization increases the binding of EGCG to serum albumin revealed by kinetic data from label-free optical biosensor with reference channel.

Epigallocatechin-gallate (EGCG) is the main polyphenol ingredient of green tea. This compound is a strong antioxidant and oxidizes easily. Numerous studies demonstrated its beneficial effects on the human health, for example its anticancer and anti-inflammatory activity. In the body, EGCG is transported by serum albumin. EGCG easily oxidizes and the interactions of the oxidized form presumably present significant differences. However, the presence of oxidized EGCG is usually neglected in the literature and its effects have not been investigated in detail. Here, we applied the label-free grating coupled interferometry method that performs dual-channel measurements. The measured kinetic signal can be compensated with a signal of a reference channel at each measurement time. By testing both hydrophilic and hydrophobic platforms, we found that EGCG can bind to a wide range of surfaces. Exploiting the dual-channel referencing ability as well as the unique sensitivity and throughput of the employed label-free technique, the experiments revealed the specific interactions between bovine serum albumin (BSA) and EGCG and determined the characteristic dissociation constant (Kd) of the binding equilibrium. The obtained binding constants were compared to literature values, showing reasonable agreement with NMR data. Besides the native EGCG, the oxidized form of EGCG was also examined, whose binding behaviors to serum albumins have never been studied. Overstoichiometric binding obtained; BSA has stronger and weaker binding sites, which could be characterized by two separate Kd values. Furthermore, EGCG oxidization increased the bound amount.

Adhesion force measurements on functionalized microbeads: An in-depth comparison of computer controlled micropipette and fluidic force microscopy.

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.

Biomimetic Dextran-Based Hydrogel Layers for Cell Micropatterning over Large Areas Using the FluidFM BOT Technology.

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.

In situ viscoelastic properties and chain conformations of heavily hydrated carboxymethyl dextran layers: a comparative study using OWLS and QCM-I chips coated with waveguide material.

Hydration, viscoelastic properties and dominant structure of thin polymer layers on the surface of waveguide material were evaluated using optical waveguide lightmode spectroscopy (OWLS) and quartz crystal microbalance (QCM) methods. The fundamentally different principles of the two applied label-free biosensors enable to examine analyte layers from complementary aspects, e.g. to determine the amount of bound water in hydrated layers. In this study, a new QCM instrument with impedance measurement (QCM-I) is introduced. Its specially designed sensor chips, covered by thin film of waveguide material, supply identical surface as used in OWLS sensors, thus enabling to perform parallel measurements on the same type of surface. Viscoelastic analysis of the measured data was performed by our evaluation code developed in MATLAB environment, using the Voinova's Voigt-based model. In situ deposition experiments on the ultrathin films of poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) were conducted for instrumental and code validation. Additionally, a novel OWLS-QCM data evaluation methodology has been developed based on the concept of combining hydration and viscoelastic data with optical anisotropy results from OWLS measurements. This methodology provided insight into the time-dependent chain conformation of heavily hydrated nano-scaled layers, resulting in unprecedented structural, hydration and viscoelastic information on covalently grafted ultrathin carboxymethyl dextran (CMD) films. The measured mass values as well as hydration and viscoelastic properties were compared with the characteristics of PLL-g-PEG layers.

Green tea polyphenol tailors cell adhesivity of RGD displaying surfaces: multicomponent models monitored optically.

The interaction of the anti-adhesive coating, poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) and its Arg-Gly-Asp (RGD) functionalized form, PLL-g-PEG-RGD, with the green tea polyphenol, epigallocatechin-gallate (EGCg) was in situ monitored. After, the kinetics of cellular adhesion on the EGCg exposed coatings were recorded in real-time. The employed plate-based waveguide biosensor is applicable to monitor small molecule binding and sensitive to sub-nanometer scale changes in cell membrane position and cell mass distribution; while detecting the signals of thousands of adhering cells. The combination of this remarkable sensitivity and throughput opens up new avenues in testing complicated models of cell-surface interactions. The systematic studies revealed that, despite the reported excellent antifouling properties of the coatings, EGCg strongly interacted with them, and affected their cell adhesivity in a concentration dependent manner. Moreover, the differences between the effects of the fresh and oxidized EGCg solutions were first demonstrated. Using a semiempirical quantumchemical method we showed that EGCg binds to the PEG chains of PLL-g-PEG-RGD and effectively blocks the RGD sites by hydrogen bonds. The calculations supported the experimental finding that the binding is stronger for the oxidative products. Our work lead to a new model of polyphenol action on cell adhesion ligand accessibility and matrix rigidity.

Fabrication and characterization of ultrathin dextran layers: Time dependent nanostructure in aqueous environments revealed by OWLS.

Surface coatings of the polysaccharide dextran and its derivatives are key ingredients especially in label-free biosensors for the suppression of non-specific binding and for receptor immobilization. Nevertheless, the nanostructure of these ultrathin coatings and its tailoring by the variation of the preparation conditions have not been profoundly characterized and understood. In this work carboxymethylated dextran (CMD) was prepared and used for fabricating ultrathin surface coatings. A grafting method based on covalent coupling to aminosilane- and epoxysilane-functionalized surfaces was applied to obtain thin CMD layers. The carboxyl moiety of the CMD was coupled to the aminated surface by EDC-NHS reagents, while CMD coupling through epoxysilane molecules was performed without any additional reagents. The surface analysis following the grafting procedures consisted of X-ray photoelectron spectroscopy (XPS), attenuated total reflection infrared spectroscopy (ATR-IR), spectroscopic ellipsometry, atomic force microscopy (AFM) and optical waveguide lightmode spectroscopy (OWLS). The XPS and AFM measurements showed that the grafting resulted in a very thin dextran layer of a few nanometers. The OWLS method allowed devising the structure of the interfacial dextran layers by the evaluation of the optogeometrical parameters. The alteration in the nanostructure of the CMD layer with the chemical composition of the silane coverage and the pH of the grafting solution was revealed by in situ OWLS, specifically, lain down chains were found to be prevalent on the surface under neutral and basic conditions on epoxysilylated surfaces. The developed methodologies allowed to design and fabricate nanometer scale CMD layers with well-controlled surface structure, which are very difficult to characterize in aqueous environments using present instrumentations and highly hydrated surface layers.

Flagellin based biomimetic coatings: From cell-repellent surfaces to highly adhesive coatings.

UNLABELLED: Biomimetic coatings with cell-adhesion-regulating functionalities are intensively researched today. For example, cell-based biosensing for drug development, biomedical implants, and tissue engineering require that the surface adhesion of living cells is well controlled. Recently, we have shown that the bacterial flagellar protein, flagellin, adsorbs through its terminal segments to hydrophobic surfaces, forming an oriented monolayer and exposing its variable D3 domain to the solution. Here, we hypothesized that this nanostructured layer is highly cell-repellent since it mimics the surface of the flagellar filaments. Moreover, we proposed flagellin as a carrier molecule to display the cell-adhesive RGD (Arg-Gly-Asp) peptide sequence and induce cell adhesion on the coated surface. The D3 domain of flagellin was replaced with one or more RGD motifs linked by various oligopeptides modulating flexibility and accessibility of the inserted segment. The obtained flagellin variants were applied to create surface coatings inducing cell adhesion and spreading to different levels, while wild-type flagellin was shown to form a surface layer with strong anti-adhesive properties. As reference surfaces synthetic polymers were applied which have anti-adhesive (PLL-g-PEG poly(l-lysine)-graft-poly(ethylene glycol)) or adhesion inducing properties (RGD-functionalized PLL-g-PEG). Quantitative adhesion data was obtained by employing optical biochips and microscopy. Cell-adhesion-regulating coatings can be simply formed on hydrophobic surfaces by using the developed flagellin-based constructs. The developed novel RGD-displaying flagellin variants can be easily obtained by bacterial production and can serve as alternatives to create cell-adhesion-regulating biomimetic coatings. STATEMENT OF SIGNIFICANCE: In the present work, we show for the first time that.

Label-free in situ optical monitoring of the adsorption of oppositely charged metal nanoparticles.

The mechanism of alternating deposition of oppositely charged gold nanoparticles (AuNPs) was investigated by optical waveguide lightmode spectroscopy (OWLS). OWLS allows monitoring of the kinetics of layer-by-layer (LbL) adsorption of positively and negatively charged nanoparticles in real time without using any labels so that the dynamics of layer formation can be revealed. Positively charged NPs that are already deposited on a negatively charged glass substrate strongly facilitate the adsorption of the negatively charged particles. The morphology of the adsorbed layer was also investigated with atomic force microscopy (AFM). AFM revealed that the interaction between oppositely charged particles results in the formation of NP clusters with sizes varying between 100 and 6000 NPs. The cluster size distribution is found to be an exponentially decaying function, and we propose a simple theory to explain this finding.

Sample handling in surface sensitive chemical and biological sensing: a practical review of basic fluidics and analyte transport.

This paper gives an overview of the advantages and associated caveats of the most common sample handling methods in surface-sensitive chemical and biological sensing. We summarize the basic theoretical and practical considerations one faces when designing and assembling the fluidic part of the sensor devices. The influence of analyte size, the use of closed and flow-through cuvettes, the importance of flow rate, tubing length and diameter, bubble traps, pressure-driven pumping, cuvette dead volumes, and sample injection systems are all discussed. Typical application areas of particular arrangements are also highlighted, such as the monitoring of cellular adhesion, biomolecule adsorption-desorption and ligand-receptor affinity binding. Our work is a practical review in the sense that for every sample handling arrangement considered we present our own experimental data and critically review our experience with the given arrangement. In the experimental part we focus on sample handling in optical waveguide lightmode spectroscopy (OWLS) measurements, but the present study is equally applicable for other biosensing technologies in which an analyte in solution is captured at a surface and its presence is monitored. Explicit attention is given to features that are expected to play an increasingly decisive role in determining the reliability of (bio)chemical sensing measurements, such as analyte transport to the sensor surface; the distorting influence of dead volumes in the fluidic system; and the appropriate sample handling of cell suspensions (e.g. their quasi-simultaneous deposition). At the appropriate places, biological aspects closely related to fluidics (e.g. cellular mechanotransduction, competitive adsorption, blood flow in veins) are also discussed, particularly with regard to their models used in biosensing.

Optical anisotropy of flagellin layers: in situ and label-free measurement of adsorbed protein orientation using OWLS.

The surface adsorption of the protein flagellin was followed in situ using optical waveguide lightmode spectroscopy (OWLS). Flagellin did not show significant adsorption on a hydrophilic waveguide, but very rapidly formed a dense monolayer on a hydrophobic (silanized) surface. The homogeneous and isotropic optical layer model, which has hitherto been generally applied in OWLS data interpretation for adsorbed protein films, failed to characterize the flagellin layer, but it could be successfully modeled as an uniaxial thin film. This anisotropic modeling revealed a significant positive birefringence in the layer, suggesting oriented protein adsorption. The adsorbed flagellin orientation was further evidenced by monitoring the surface adsorption of truncated flagellin variants, in which the terminal protein regions or the central (D3) domain were removed. Without the terminal regions the protein adsorption was much slower and the resulting films were significantly less birefringent, implying that intact flagellin adsorbs on the hydrophobic surface via its terminal regions.

Self-assembly of rodlike receptors from bulk solution.

Optical waveguide lightmode spectroscopy has been used to observe the deposition of bacterial flagellar filaments of mean length 350 nm from bulk solution onto a smooth planar substratum, chemically modified to covalently bind the flagellar filaments on contact. At the highest practicable bulk concentration, the filaments follow the theoretically predicted kinetics of random sequential addition of highly elongated rigid rods to the substratum, but addition terminates with the rods almost perpendicular to the substratum. Rod-rod correlations in the bulk anomalously accelerate the rate of arrival of the filaments at the surface of the substratum, relative to spheres. At lower concentrations, this effect is absent, and the rods have time to order themselves on the substratum, forming a two-dimensional array.