Inna Székács

Optical waveguide light-mode spectroscopy immunosensors for environmental monitoring

Coupling the high specificity of the immunoanalytical reaction with the high sensitivity of optical waveguide light-mode spectroscopy (OWLS) detection gives the possibility to develop immunosensors with in most cases a definitely lower detection limit than traditionally used immunoassays. Measurements were performed on the sensitized surface of optical waveguide grating coupler sensors (2400 lines/mm grating). The OWLS technique is based on the precise measurement of the resonance angle of a polarized laser light (632.8 nm), diffracted by a grating and incoupled into a thin waveguide. The effective refractive index, determined from the resonance incoupling angle detected at high accuracy, allows determination of layer thickness and coverage (or mass) of the adsorbed or bound material with ultrahigh sensitivity. OWLS immunosensors were developed as label-free immunosensors with an amino group modified SiO(2)-TiO(2) sensor surface on which the immunoreactants could be anchored. One of the components of the antibody-antigen complex was chemically bound on the sensor surface, allowing noncompetitive or competitive detection of the analytes. To illustrate that the resulting immunosensors are suitable for the determination of small and large molecular weight analytes, OWLS sensor formats were applied for quantitative detection of a herbicide active ingredient trifluralin, a Fusarium mycotoxin zearalenone, and an egg yolk protein of key importance in endocrine regulation, vitellogenin.

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.

Incubator proof miniaturized Holomonitor to in situ monitor cancer cells exposed to green tea polyphenol and preosteoblast cells adhering on nanostructured titanate surfaces: validity of the measured parameters and their corrections

Abstract. The in situ observation of cell movements and morphological parameters over longer periods of time under physiological conditions is critical in basic cell research and biomedical applications. The quantitative phase-contrast microscope applied in this study has a remarkably small size, therefore it can be placed directly into a humidified incubator. Here, we report on the successful application of this M4 Holomonitor to observe cancer cell motility, motility speed, and migration in the presence of the green tea polyphenol, epigallocatechin gallate, as well as to monitor the adhesion of preosteoblast cells on nanostructured titanate coatings, relevant for biomedical applications. A special mechanical stage was developed to position the sample into that range of the optical arrangement where digital autofocusing works with high reproducibility and precision. By in-depth analyzing the obtained single cell morphological parameters, we show that the limited vertical resolution of the optical setup results in underestimated single cell contact area and volume and overestimated single cell averaged thickness. We propose a simple model to correct the recorded data to obtain more precise single cell parameters. We compare the results with the kinetic data recorded by a surface sensitive optical biosensor, optical waveguide lightmode spectroscopy.

Plasmon-enhanced two-channel in situ Kretschmann ellipsometry of protein adsorption, cellular adhesion and polyelectrolyte deposition on titania nanostructures

Plasmon-enhanced in situ spectroscopic ellipsometry was realized using the Kretschmann geometry. A 10-μL flow cell was designed for multi-channel measurements using a semi-cylindrical lens. Dual-channel monitoring of the layer formation of different organic structures has been demonstrated on titania nanoparticle thin films supported by gold. Complex modeling capabilities as well as a sensitivity of ~40 pg/mm2 with a time resolution of 1 s was achieved. The surface adsorption was enhanced by the titania nanoparticles due to the larger specific surface and nanoroughness, which is consistent with our previous results on titanate nanotubes.

Label-free optical biosensor for real-time monitoring the cytotoxicity of xenobiotics: A proof of principle study on glyphosate

Rapid and inexpensive biosensor technologies allowing real-time analysis of biomolecular and cellular events have become the basis of next-generation cell-based screening techniques. Our work opens up novel opportunities in the application of the high-throughput label-free Epic BenchTop optical biosensor in cell toxicity studies. The Epic technology records integrated cellular responses about changes in cell morphology and dynamic mass redistribution of cellular contents at the 100-150 nm layer above the sensor surface. The aim of the present study was to apply this novel technology to identify the effect of the herbicide Roundup Classic, its co-formulant polyethoxylated tallow amine (POEA), and its active ingredient glyphosate, on MC3T3-E1 cells adhered on the biosensor surface. The half maximal inhibitory concentrations of Roundup Classic, POEA and glyphosate upon 1 h of exposure were found to be 0.024%, 0.021% and 0.163% in serum-containing medium and 0.028%, 0.019% and 0.538% in serum-free conditions, respectively (at concentrations equivalent to the diluted Roundup solution). These results showed a good correlation with parallel end-point assays, demonstrating the outstanding utility of the Epic technique in cytotoxicity screening, allowing not only high-throughput, real-time detection, but also reduced assay run time and cytotoxicity assessment at end-points far before cell death would occur.

ZnO Nanostructure Templates as a Cost-Efficient Mass-Producible Route for the Development of Cellular Networks

The development of artificial surfaces which can regulate or trigger specific functions of living cells, and which are capable of inducing in vivo-like cell behaviors under in vitro conditions has been a long-sought goal over the past twenty years. In this work, an alternative, facile and cost-efficient method for mass-producible cellular templates is presented. The proposed methodology consists of a cost-efficient, two-step, all-wet technique capable of producing ZnO-based nanostructures on predefined patterns on a variety of substrates. ZnO—apart from the fact that it is a biocompatible material—was chosen because of its multifunctional nature which has rendered it a versatile material employed in a wide range of applications. Si, Si3N4, emulated microelectrode arrays and conventional glass cover slips were patterned at the micrometer scale and the patterns were filled with ZnO nanostructures. Using HeLa cells, we demonstrated that the fabricated nanotopographical features could promote guided cellular adhesion on the pre-defined micron-scale patterns only through nanomechanical cues without the need for further surface activation or modification. The basic steps of the micro/nanofabrication are presented and the results from the cell adhesion experiments are discussed, showing the potential of the suggested methodology for creating low-cost templates for engineered cellular networks.

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.

Determination of histamine content in vegetable juices by using direct and competitive immunosensors

An Optical Waveguide Lightmode Spectroscopy (OWLS)-based immunosensor was developed for selective and sensitive determination of histamine content in fermented vegetable juices. The conjugate of the antigen (histamine-bovine serum albumin 10 µg mL−1) was attached on the amino modified sensor surface with glutaraldehyde. During the measurement standard solutions or samples were mixed with antibodies of appropriate concentration (1:1000), the mixture was incubated for 1 minute and injected into the OWLS system. The amount of antibodies bound to immobilised antigen conjugates was inversely proportional to the histamine content. Relative substrate specificity of the antibody was studied, and besides histamine, only putrescine, cadaverine and agmatine were found to give slight sensor responses. Fermented vegetable juice samples treated with different microorganisms were measured in 1000-fold dilution, and analytical data were compared to those measured by HPLC. It was concluded that the method is suitable for the sensitive and selective determination of histamine content.

Optical Waveguide Lightmode Spectroscopic Techniques for Investigating Membrane-Bound Ion Channel Activities

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.

Label-Free Optical Biosensors for Monitoring Cellular Processes and Cytotoxic Agents at Interfaces Using Guided Modes and Advanced Phase-Contrast Imaging Techniques

Novel optical biosensors in direct interaction with living cells open new avenues in the detection of given cell types, including pathogenic microorganisms, and in the measurement of cytotoxicity of given xenobiotics, such as biothreat agents, on suitably selected cell types or lines. Evanescent optical field based biosensors emerging for such applications are reviewed, covering surface plasmon and waveguide based formats, imaging setup, employing optical waveguides, and digital holographic microscopy. Commercial technologies, along with main pathogenic microorganisms (Bacillus anthracis, Escherichia coli, Listeria monocytogenes, Campylobacter jejuni, etc.) as analytes and cell lines (native tissue and tumor cells) as cytotoxicity effect subjects are highlighted.