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.
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.
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•Kinetic and structural information of nanolayers revealed by label-free biosensors•Classical and novel label-free methods and their combinations for complex analysis•Adsorption kinetics: theory and modeling experimental conditions•Practical overview on the effect of model parameters on kinetic fits•Software packages for QCM and OWLS data evaluation as well as for kinetic analysis
Tissue-on-a-chip technologies are more and more important in the investigation of cellular function and in the development of novel drugs by allowing the direct screening of substances on human ...cells. Constituting the inner lining of vessel walls, endothelial cells are the key players in various physiological processes, moreover, they are the first to be exposed to most drugs currently used. However, to date, there is still no appropriate technology for the label-free, real-time and high-throughput monitoring of endothelial function. To this end, we developed an optical biosensor-based endothelial label-free biochip (EnLaB) assay that meets all the above requirements. Using our EnLaB platform, we screened a set of plasma serine proteases as possible endothelial cell activators, and first identified the endothelial cell activating function of three important serine proteases - namely kallikrein, C1r and mannan-binding lectin-associated serine-protease 2 (MASP-2) - and verified these results in well-established functional assays. EnLaB proved to be an effective tool for revealing novel cellular mechanisms as well as for the high-throughput screening of various compounds on endothelial cells.
Single-cell adhesion force plays a crucial role in biological sciences, however its in-depth investigation is hindered by the extremely low throughput and the lack of temporal resolution of present ...techniques. While atomic force microcopy (AFM) based methods are capable of directly measuring the detachment force values between individual cells and a substrate, their throughput is limited to few cells per day, and cannot provide the kinetic evaluation of the adhesion force over the timescale of several hours. In this study a high spatial and temporal resolution resonant waveguide grating based label-free optical biosensor was combined with robotic fluidic force microscopy to monitor the adhesion of living cancer cells. In contrast to traditional fluidic force microscopy methods with a manipulation range in the order of 300-400 micrometers, the robotic device employed here can address single cells over mm-cm scale areas. This feature significantly increased measurement throughput, and opened the way to combine the technology with the employed microplate-based, large area biosensor. After calibrating the biosensor signals with the direct force measuring technology on 30 individual cells, the kinetic evaluation of the adhesion force and energy of large cell populations was performed for the first time. We concluded that the distribution of the single-cell adhesion force and energy can be fitted by log-normal functions as cells are spreading on the surface and revealed the dynamic changes in these distributions. The present methodology opens the way for the quantitative assessment of the kinetics of single-cell adhesion force and energy with an unprecedented throughput and time resolution, in a completely non-invasive manner.
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.
•We summarize the current state of methods for label-free identification and functional characterization of leukocytes•Labeling cells might affect their behavior, and therefore lead to a biased description of the investigated phenomena•Label-free biosensors can decrease processing time, reagent costs and enable point-of-care diagnostics•Label-free detection allows real-time monitoring of kinetic processes, and enable downstream investigation of cells•Surface sensitive technologies enable the characterizations of single cells for diagnostic purposes•Techniques capable of analyzing single cells are promising tools for therapeutic approaches as well
A phase shifting optical waveguide interferometer is demonstrated for label-free evanescent wave biosensing in aqueous solutions. In the proposed configuration, the reference and measurement arms of ...the interferometer are combined inside an integrated optical Ta
2O
5 waveguide using an ion implanted grating. Biomolecules adsorbing on the waveguide surface shift the phase in the measurement arm, which is modulated at the same time by a periodically relaxing liquid crystal phase modulator. It is demonstrated that by analyzing the periodic intensity response at the end facet of the waveguide, the phase shifts in the measurement arm can be monitored in real-time with a precision of 10
−4
rad. The high phase resolution allows the detection of surface adsorbed molecule densities below 1
pg/mm
2 without using any labeling or on-chip referencing. The instrument performance is demonstrated by monitoring in situ protein adsorption and affinity binding of low molecular weight pure biotin to an immobilized avidin layer.
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.
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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.
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.
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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.
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•Bio sensing often suffers from inappropriate fluidic design/sample handling.•We overview the basic theory of fluidics and that of analyte transport.•We compare various fluidic setups from a practical point of view.•Effects of dead volumes, tubing length, flow, etc. are all illustrated with OWLS data.•We highlight the biological aspects of fluid handling (e.g. cell behavior in flow).
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