Összes szerző

Kurunczi Sándor

az alábbi absztraktok szerzői között szerepel:

Farkas Enikő
Controlling Live Cell Adhesion through Characterization of Biofunctionalized Surfaces using Label-Free Biosensors

Aug 30 - szerda

15:30 – 17:00

II. Poszterszekció

P41

Controlling Live Cell Adhesion through Characterization of Biofunctionalized Surfaces using Label-Free Biosensors

Eniko Farkas¹, Kinga Dóra Kovács^1,3, Beatrix Peter¹, Attila Bonyár², Sandor Kurunczi¹, Inna Szekacs¹, and Robert Horvath¹

¹ Nanobiosensorics Laboratory, Institute of Technical Physics and Materials Science, Centre for Energy Research, Budapest, Hungary

² Department of Electronics Technology, Faculty of Electrical Engineering and Informatics, Budapest University of Technology and Economics, Budapest, Hungary

Biomaterial coatings that possess cell-repellent or cell-adhesive properties have a significant interest in medical and biotechnological applications [1-4]. However, conventional approaches lack in-depth analysis and quantitative comparison of these coatings for regulating adhesion, particularly for bacterial cell adhesion. Label-free Optical Waveguide Lightmode Spectroscopy (OWLS) can offer a solution for the detailed analysis of biomaterial coatings. OWLS biosensors detect the optical properties of the adhesive surface using evanescent waves with a penetration depth of 100-150 nm [5-7]. This surface-sensitive technique enables a thorough evaluation of biomaterial coatings for regulating adhesion. Uniquely, OWLS enables the in situ measurement of both the coating process and subsequent cell adhesion.

The present study utilizes the OWLS method for in-depth characterization of biomaterial surfaces with regard to bacterial adhesion. Initially, adhesion blocking biomaterials, namely bovine serum albumin, I-block, PAcrAM-g-(PMOXA, NH2, Si), (PAcrAM-P), and PLL-g-PEG, with varying coating temperatures, were screened. PAcrAM-P exhibited the best blocking capability against bacterial concentrations up to 10⁷ cells/mL. Subsequently, different immobilization methods, such as Mix&Go (AnteoBind) films, protein A, avidin-biotin based surface chemistries, and simple physisorption, were employed to captureEscherichia coli specific antibodies. Bacterial cell adhesion was then tested on immobilized antibodies with various blocking agents. The OWLS analysis allowed for the determination of the parameters of the applied agents by considering the kinetic data of adhesion, the surface mass density, and the protein orientation. Based on the experimental results, surfaces were created and tested for controlling both bacterial and mammalian cell adhesion. [8]

Acknowledgment

This work was supported by the "Lendület" (HAS) research program, the National Research, Development and Innovation Office of Hungary ((ERC_HU, VEKOP 2.2.1-16, ELKH topic-fund, "Élvonal" KKP_19 and KH grants, PD 131543 and TKP2022-EGA-04 –INBIOM TKP Programs financed from the NRDI Fund). This work was also supported by 77 Elektronika Ltd. by their supplying of antibodies and reagents.

References

[1] Frutiger A, et. al. (2021) Chem Rev 121: 8095–8160.

[2] Rigo S, et. al. (2018) Adv Sci 5: 1700892.

[3] Castillo-Henríquez L, et. al. (2020) Sensors 20: 6926.

[4] D’Agata R, et. al. (2021) Polymers 13:1929.

[5] Vörös J, et. al. (2002) Biomaterials 23: 3699–3710.

[6] Tiefenthaler K, et. al. (1989) J Opt Soc Am. B 6: 209–220.

[7] Saftics A, et. al. (2021) Adv Colloid Interface Sci 294: 102431–102433.

[8] Farkas E, et. al. (2022) Biosensors 12: 56.

Kovács Boglárka
Flagellin: a convenient protein in biosensorics

Aug 30 - szerda

11:15 – 11:30

Bioszenzorika és bio-nanotechnológia

E27

Flagellin: a convenient protein in biosensorics

Boglárka Kovács¹, András Saftics¹, Inna Székács¹, Hajnalka Jankovics², Sandor Kurunczi¹, Ferenc Vonderviszt², Robert Horvath¹

¹Nanobiosensorics Laboratory, Centre for Energy Research, Institute of Technical Physics and Materials Science, Budapest, Hungary

²Bio-nanosystem Laboratory, Research Institute of Biomolecular and Chemical Engineering, University of Pannonia, Veszprém, Hungary

Flagellin is the main building block of bacterial flagellar filaments. Since the filaments are located outside of the cells, cell lysis is not required to purify flagellin. Flagellin consists of 4 domains: D0, D1, D2, and D3. The D0 domain contains amphipathic helical regions with hydrophobic amino acids on one side of the helix. This part of flagellin is disordered in solution, but can be used to anchor the protein on hydrophobic surfaces with the D3 domain pointing towards the solution [1]. The hypervariable D3 domain situated on the filament surface is a largely independent part of the flagellin that can be removed or replaced without disturbing filament formation.

During our work we in-depth characterized the coatings created from flagellin, and influenced the adsorption of the protein with Hofmeister salts [1]. We applied genetically modified high affinity Ni-binding variant as receptor, and demonstrated the unique sensitivity of grating-coupled interferometry [2].

The monolayer of wild-type flagellin mimics the surface of the bacterial flagellar filament, and we hypothesized that oriented flagellin layers have bacteria-repellent properties. To prove this, we studied the adhesion of bacterial E. coli and human cancer cells on oriented wild-type flagellin layers [3,4].

Through genetic modification, specific oligopeptide segments can be also inserted into the D3 domain of flagellin, which can induce cell adhesion through integrin receptors. We studied cancer cell adhesion on the genetically engineered protein layers with label-free optical biosensors [4]. Mammalian cells can recognize flagellin in solution through Toll-like receptors, and the protein can cause innate immune system response. We are studying the above biological mechanisms and its consequences in the adhesion of the flagelljn exposed cells. Our results prove, that flagellin can be used in many ways in creating capture layers in biosensors.

References

[1] Kovacs, B.; Saftics, A.; Biro, A.; Kurunczi, S.; Szalontai, B.; Kakasi, B.; Vonderviszt, F.; Der, A.; Horvath, R. J. Phys. Chem. C 2018, 122 (37), 21375–21386.

[2] Jankovics, H.; Kovacs, B.; Saftics, A.; Gerecsei, T.; Tóth, É.; Szekacs, I.; Vonderviszt, F.; Horvath, R. Sci. Rep. 2020, 1–11.

[3] Kovacs, B.; Patko, D.; Klein, A.; Kakasi, B.; Saftics, A.; Kurunczi, S.; Vonderviszt, F.; Horvath, R. Sensors Actuators B Chem. 2018, 257, 839–845.

[4] Kovacs, B.; Patko, D.; Szekacs, I.; Orgovan, N.; Kurunczi, S.; Sulyok, A.; Khanh, N. Q.; Toth, B.; Vonderviszt, F.; Horvath, R. Acta Biomater. 2016, No. 42, 66–76.

Kovács Dóra Kinga
Nanoinjection of fluorescent nanoparticles to single live cells by robotic fluidic force microscopy

Aug 29 - kedd

12:15 – 12:30

Modern biofizikai módszerek

E13

Nanoinjection of fluorescent nanoparticles to single live cells by robotic fluidic force microscopy

Kinga Dóra Kovács^1,*, Tamás Visnovitz^2,3,*, Tamás Gerecsei¹, Beatrix Peter¹, Sándor Kurunczi¹, Anna Koncz², Krisztina Németh², Dorina Lenzinger², Krisztina V. Vukman², Péter Lőrincz⁴, Inna Székács¹, Edit I. Buzás^2,5,6**, Robert Horvath^1,**

¹ Nanobiosensorics Laboratory, Centre of Energy Research, ELKH, Budapest, Hungary

² Department of Genetics, Cell- and Immunobiology, Semmelweis University, Budapest, Hungary

³ Department of Plant Physiology and Molecular Plant Biology, ELTE Eötvös Loránd University, Budapest, Hungary

⁴ Department of Anatomy, Cell and Developmental Biology, ELTE Eötvös Loránd University, Budapest, Hungary

⁵ HCEMM-SU Extracellular Vesicle Research Group, Budapest, Hungary

⁶ ELKH-SE Translational Extracellular Vesicle Research Group, Budapest, Hungary

*,** equal contributions / **corresponding authors

Direct injection of fluorescent nanoparticles into the cytoplasm of living cells can provide new insights into the intracellular fate of various different fluorescently labelled biologically active particles. Here we used fluorescent nanoparticles to prove the feasibility of nanoinjection into single live HeLa cells by using robotic fluidic force microscopy (FluidFM). This injection platform offers the advantage of high cell selectivity and efficiency. We confirmed the successful injection of both GFP encoding plasmids and GFP tagged fluorescent nanoparticles to the cells by confocal microscopy. We were able track the nanoparticles in the living cells for 20 hours. The injected nanoparticles 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, the FluidFM platform is suitable for targeting single living cells by fluorescently labelled biologically active particles and may lead to information about the intracellular cargo delivery at a single-cell level.