Sándor Kurunczi

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.

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.

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.

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.

Design and process development of a photonic crystal polymer biosensor for point-of-care diagnostics

In this work, we report advances in the fabrication and anticipated performance of a polymer biosensor photonic chip developed in the European Union project P3SENS (FP7-ICT4-248304). Due to the low cost requirements of point-ofcare applications, the photonic chip is fabricated from nanocomposite polymeric materials, using highly scalable nanoimprint- lithography (NIL). A suitable microfluidic structure transporting the analyte solutions to the sensor area is also fabricated in polymer and adequately bonded to the photonic chip. We first discuss the design and the simulated performance of a high-Q resonant cavity photonic crystal sensor made of a high refractive index polyimide core waveguide on a low index polymer cladding. We then report the advances in doped and undoped polymer thin film processing and characterization for fabricating the photonic sensor chip. Finally the development of the microfluidic chip is presented in details, including the characterisation of the fluidic behaviour, the technological and material aspects of the 3D polymer structuring and the stable adhesion strategies for bonding the fluidic and the photonic chips, with regards to the constraints imposed by the bioreceptors supposedly already present on the sensors.

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.

NIL fabrication of a polymer-based photonic sensor device in P3SENS project

We present the most recent results of EU funded project P3SENS (FP7-ICT-2009.3.8) aimed at the development of a low-cost and medium sensitivity polymer based photonic biosensor for point of care applications in proteomics. The fabrication of the polymer photonic chip (biosensor) using thermal nanoimprint lithography (NIL) is described. This technique offers the potential for very large production at reduced cost. However several technical challenges arise due to the properties of the used materials. We believe that, once the NIL technique has been optimised to the specific materials, it could be even transferred to a kind of roll-to-roll production for manufacturing a very large number of photonic devices at reduced cost.

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.

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.