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Characterization of carotid endothelial cell proliferation on Au, Au/GO, and Au/rGO surfaces by electrical impedance spectroscopy

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Abstract

To the best of the authors’ knowledge, testing the biocompatibility of graphene coatings can be considered as the first to demonstrate human carotid endothelial cell (HCtAEC) proliferation on Au, graphene oxide–coated Au (Au/GO), and reduced graphene oxide–coated Au (Au/rGO) surfaces. We hypothesized that stent material modified with graphene (G)-based coatings could be used as electrodes for electrical impedance spectroscopy (EIS) in monitoring cell cultures, i.e., endothelialization. Alamar Blue cell viability assay and cell staining and cell counting with optical images were performed. For EIS analysis, an EIS sensor consisting of Au surface electrodes was produced by the photolithographic technique. Surface characterizations were performed by considering scanning electron microscope (SEM) and water contact angle analyses. Results showed that GO and rGO coatings did not prevent neither the electrical measurements nor the cell proliferation and that rGO had a positive effect on HCtAEC proliferation. The rate of increase of impedance change from day 1 to day 10 was nearly fivefold for all electrode surfaces. Alamar Blue assay performed to monitor cell proliferation rates between groups, and rGO has shown the highest Alamar Blue reduction value of 43.65 ± 8.79%.

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References

  1. Bedair TM, ElNaggar MA, Joung YK, Han DK (2017) Recent advances to accelerate re-endothelialization for vascular stents. J Tissue Eng 8:204173141773154

    Google Scholar 

  2. Zhang K, Liu T, Li J, Chen J, Wang J, Huang N (2013) Surface modification of implanted cardiovascular metal stents : from antithrombosis and antirestenosis to endothelialization. J Biomed Mater Res A 102A:588–609

    Google Scholar 

  3. Curcio A, Torella D, Indolfi C (2011) Mechanisms of smooth muscle cell proliferation and endothelial regeneration after vascular injury and stenting. Circ J 75(6):1287–1296

    CAS  PubMed  Google Scholar 

  4. Tan CH, Muhamad N, Abdullah MMAB (2017) Surface topographical modification of coronary stent: a review. IOP Conf Ser Mater Sci Eng 209(012031):1–9

  5. Xu X et al (2017) Polymer coating embolism from intravascular medical devices — a clinical literature review. J Biomater Appl 114(1):18L–21L

    Google Scholar 

  6. Li G, Yang P, Qin W, Maitz MF, Zhou S, Huang N (2011) The effect of coimmobilizing heparin and fibronectin on titanium on hemocompatibility and endothelialization. Biomaterials 32(21):4691–4703

    CAS  PubMed  Google Scholar 

  7. Chen Y, Shayan M, Yeo WH, Chun Y (2017) Assessment of endothelial cell growth behavior in thin film nitinol. Biochip J 11(1):39–45

    CAS  Google Scholar 

  8. Liu Y et al (Sep. 2014) Tailoring of the dopamine coated surface with VEGF loaded heparin/poly-l-lysine particles for anticoagulation and accelerate in situ endothelialization. J Biomed Mater Res A:1–11

  9. Buccheri D, Piraino D, Andolina G, Cortese B (2016) Understanding and managing in-stent restenosis: a review of clinical data, from pathogenesis to treatment. J Thorac Dis 8(10):E1150–E1162

    PubMed  PubMed Central  Google Scholar 

  10. Rebagay G, Bangalore S (2019) Biodegradable polymers and stents: the next generation? Curr Cardiovasc Risk Rep 13(8):22

    Google Scholar 

  11. Ge S et al. (2019) Inhibition of in-stent restenosis after graphene oxide double-layer drug coating with good biocompatibility. Regen Biomater Oct 6(5):299–309

  12. Cardenas L, MacLeod J, Lipton-Duffin J, Seifu DG, Popescu F, Siaj M, Mantovani D, Rosei F (2014) Reduced graphene oxide growth on 316L stainless steel for medical applications. Nanoscale 6(15):8664–8670

    CAS  PubMed  Google Scholar 

  13. Podila R, Moore T, Alexis F, Rao AM (2013) Graphene coatings for enhanced hemo-compatibility of nitinol stents. RSC Adv 3(6):1660

    CAS  Google Scholar 

  14. Pan CJ, Pang LQ, Gao F, Wang YN, Liu T, Ye W, Hou YH (2016) Anticoagulation and endothelial cell behaviors of heparin-loaded graphene oxide coating on titanium surface. Mater Sci Eng C 63:333–340

    CAS  Google Scholar 

  15. Shedden L, Kennedy S, Wadsworth R, Connolly P (2010) Towards a self-reporting coronary artery stent--measuring neointimal growth associated with in-stent restenosis using electrical impedance techniques. Biosens Bioelectron 26(2):661–666

    CAS  PubMed  Google Scholar 

  16. Occhiuzzi C, Contri G, Marrocco G (2012) Design of implanted RFID tags for passive sensing of human body: the STENTag. IEEE Trans Antennas Propag 60(7):3146–3154

    Google Scholar 

  17. Anh-Nguyen T, Tiberius B, Pliquett U, Urban GA (2015) An impedance biosensor for monitoring cancer cell attachment, spreading and drug-induced apoptosis. Sensors Actuators A Phys 241:231–237

    Google Scholar 

  18. Yi-Yu L, Ji-Jer H, Yu-Jie H, Kuo-Sheng C (2013) Cell growth characterization using multi-electrode bioimpedance spectroscopy. Meas Sci Technol 24(3):35701

    Google Scholar 

  19. Cho S, Thielecke H (2008) Electrical characterization of human mesenchymal stem cell growth on microelectrode. Microelectron Eng 85(5–6):1272–1274

    CAS  Google Scholar 

  20. Qiu Y, Liao R, Zhang X (2009) Impedance-based monitoring of ongoing cardiomyocyte death induced by tumor necrosis factor-alpha. Biophys J 96(5):1985–1991

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Pradhan R, Rajput S, Mandal M, Mitra A, Das S (2014) Frequency dependent impedimetric cytotoxic evaluation of anticancer drug on breast cancer cell. Biosens Bioelectron 55:44–50

    CAS  PubMed  Google Scholar 

  22. Yun Y, Dong Z, Tan Z, Schulz MJ (2010) Development of an electrode cell impedance method to measure osteoblast cell activity in magnesium-conditioned media. Anal Bioanal Chem 396(8):3009–3015

    CAS  PubMed  Google Scholar 

  23. Mansor AFM, Ibrahim I, Zainuddin AA, Voiculescu I, Nordin AN (2017) Modeling and development of screen-printed impedance biosensor for cytotoxicity studies of lung carcinoma cells. Med Biol Eng Comput:1–9

  24. Gelsinger ML, Tupper LL, Matteson DS (2017) Cell line classification using electric cell-substrate impedance sensing (ECIS). Int J Biostat. https://doi.org/10.1515/ijb-2018-0083

  25. Wang X et al (2018) Three-dimensional graphene biointerface with extremely high sensitivity to single cancer cell monitoring. Biosens Bioelectron 105(928):22–28

    CAS  PubMed  Google Scholar 

  26. Liu Q et al. (2009) Impedance studies of bio-behavior and chemosensitivity of cancer cells by micro-electrode arrays. Biosens Bioelectron 24(5):1305–1310

  27. Franks W, Schenker I, Schmutz P, Hierlemann A (2005) Impedance characterization and modeling of electrodes for biomedical applications. IEEE Trans Biomed Eng 52(7):1295–1302

    PubMed  Google Scholar 

  28. Lanche R et al (2015) Graphite oxide multilayers for device fabrication: enzyme-based electrical sensing of glucose. Phys Status Solidi Appl Mater Sci 212(6):1335–1341

    CAS  Google Scholar 

  29. Zhang D, Zhang Y, Zheng L, Zhan Y, He L (Apr. 2013) Graphene oxide/poly-L-lysine assembled layer for adhesion and electrochemical impedance detection of leukemia K562 cancer cells. Biosens Bioelectron 42:112–118

    CAS  PubMed  Google Scholar 

  30. Flaherty ML, Kissela B, Khoury JC, Alwell K, Moomaw CJ, Woo D, Khatri P, Ferioli S, Adeoye O, Broderick JP, Kleindorfer D (2012) Carotid artery stenosis as a cause of stroke. Neuroepidemiology 40(1):36–41

    PubMed  PubMed Central  Google Scholar 

  31. LayoutEditor the universal editor for GDSII, OpenAccess, OASIS... | LayoutEditor. [Online]. Available: https://layouteditor.com/. [Accessed: 20-Sep-2019]

  32. King DE (1995) Oxidation of gold by ultraviolet light and ozone at 25 °C. J Vac Sci Technol A Vacuum, Surfaces, Film 13(3):1247–1253

    CAS  Google Scholar 

  33. Ron H, Matlis S, Rubinstein I (1998) Self-assembled monolayers on oxidized metals. 2. Gold surface oxidative pretreatment, monolayer properties, and depression formation. Langmuir 14(5):1116–1121

    CAS  Google Scholar 

  34. Woodward JT, Walker ML, Meuse CW, Vanderah DJ, Poirier GE, Plant AL (2000) Effect of an oxidized gold substrate on alkanethiol self-assembly. Langmuir 16(12):5347–5353

    Google Scholar 

  35. Slaughter GE, Hobson R (2009) An impedimetric biosensor based on PC 12 cells for the monitoring of exogenous agents. Biosens Bioelectron 24(5):1153–1158

    CAS  PubMed  Google Scholar 

  36. Xu C, Yuan R, Wang X (2014) Selective reduction of graphene oxide. Carbon N Y 71(1):345

    CAS  Google Scholar 

  37. HCtAEC | Human Carotid Artery Endothelial Cells | Quality Primary Cells | Cell Applications. [Online]. Available: https://www.cellapplications.com/human-carotid-artery-endothelial-cells-hctaec. [Accessed: 13-Feb-2020]

  38. Dapi, DAPI Protocol for Fluorescence Imaging | Thermo Fisher Scientific - TR. [Online]. Available: https://www.thermofisher.com/tr/en/home/references/protocols/cell-and-tissue-analysis/protocols/dapi-imaging-protocol.html. [Accessed: 13-Feb-2020]

  39. Actin, Actin Staining Protocol | Thermo Fisher Scientific - TR. [Online]. Available: https://www.thermofisher.com/tr/en/home/references/protocols/cell-and-tissue-analysis/microscopy-protocol/actin-staining-protocol.html. [Accessed: 13-Feb-2020]

  40. Neirynck P, Schimer J, Jonkheijm P, Milroy LG, Cigler P, Brunsveld L (2015) Carborane-β-cyclodextrin complexes as a supramolecular connector for bioactive surfaces. J Mater Chem B 3(4):539–545

    CAS  PubMed  Google Scholar 

  41. Yu HZ, Zhao JW, Wang YQ, Cai SM, Liu ZF (1997) Fabricating an azobenzene self-assembled monolayer via step-by-step surface modification of a cysteamine monolayer on gold. J Electroanal Chem 438(1–2):221–224

    CAS  Google Scholar 

  42. Xu Q, Cheng H, Lehr J, Patil AV, Davis JJ (2015) Graphene oxide interfaces in serum based autoantibody quantification. Anal Chem 87:346–350

  43. Robinson JT, Perkins FK, Snow ES, Wei Z, Sheehan PE (2008) Reduced graphene oxide molecular sensors. Nano Lett 8(10):3137–3140

    CAS  PubMed  Google Scholar 

  44. Lee JS, Yoon JC, Jang JH (2013) A route towards superhydrophobic graphene surfaces: surface-treated reduced graphene oxide spheres. J Mater Chem A 1(25):7312–7315

    CAS  Google Scholar 

  45. Wegener J, Keese CR, Giaever I (2000) Electric cell-substrate impedance sensing (ECIS) as a noninvasive means to monitor the kinetics of cell spreading to artificial surfaces. Exp Cell Res 259(1):158–166

    CAS  PubMed  Google Scholar 

  46. Giaever I, Keese CR (1993) A morphological biosensor for mammalian cells. Nature 366:591–592

    CAS  PubMed  Google Scholar 

  47. Giaever I, Keese CR (1991) Micromotion of mammalian cells measured electrically. Proc Natl Acad Sci U S A 88(17):7896–7900

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Zhang X, Wang W, Li F, Voiculescu I (2017) Stretchable impedance sensor for mammalian cell proliferation measurements. Lab Chip 17(12):2054–2066

    CAS  PubMed  Google Scholar 

  49. Zhang F, Jin T, Hu Q, He P (2018) Distinguishing skin cancer cells and normal cells using electrical impedance spectroscopy. J Electroanal Chem 823(2017):531–536

    CAS  Google Scholar 

  50. Arias LR, Perry CA, Yang L (2010) Real-time electrical impedance detection of cellular activities of oral cancer cells. Biosens Bioelectron 25(10):2225–2231

    CAS  PubMed  Google Scholar 

  51. Lanche R, Pachauri V, Koppenhöfer D, Wagner P, Ingebrandt S (2014) Reduced graphene oxide-based sensing platform for electric cell-substrate impedance sensing. Phys Status Solidi 211(6):1404–1409

    CAS  Google Scholar 

  52. Chinnadayyala SR, Park J, Choi Y, Han JH, Yagati AK, Cho S (2019) Electrochemical impedance characterization of cell growth on reduced graphene oxide-gold nanoparticles electrodeposited on indium tin oxide electrodes. Appl Sci 9(2)

  53. Mishima K, Mimura A, Takahara Y, Asami K, Hanai T (1991) On-line monitoring of cell concentrations by dielectric measurements. J Ferment Bioeng 72(4):291–295

    CAS  Google Scholar 

  54. Lo CM, Keese CR, Giaever I (1995) Impedance analysis of MDCK cells measured by electric cell-substrate impedance sensing. Biophys J 69(6):2800–2807

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Das S et al (2013) Oxygenated functional group density on graphene oxide: its effect on cell toxicity. Part Part Syst Charact 30(2):148–157

    CAS  Google Scholar 

  56. Gies V, Zou S (2018) Systematic toxicity investigation of graphene oxide: evaluation of assay selection, cell type, exposure period and flake size. Toxicol Res (Camb) 7(1):93–101

    CAS  Google Scholar 

  57. Gurunathan S et al (2019) Evaluation of graphene oxide induced cellular toxicity and transcriptome analysis in human embryonic kidney cells. Nanomaterials 9(7):969

    CAS  PubMed Central  Google Scholar 

  58. Contreras-Torres FF, Rodríguez-Galván A, Guerrero-Beltrán CE, Martínez-Lorán E, Vázquez-Garza E, Ornelas-Soto N, García-Rivas G (Apr. 2017) Differential cytotoxicity and internalization of graphene family nanomaterials in myocardial cells. Mater Sci Eng C 73:633–642

    CAS  Google Scholar 

  59. Paszek E, Czyz J, Woźnicka O, Jakubiak D, Wojnarowicz J, Łojkowski W, Stepień E (2012) Zinc oxide nanoparticles impair the integrity of human umbilical vein endothelial cell monolayer in vitro. J Biomed Nanotechnol 8(6):957–967

    CAS  PubMed  Google Scholar 

  60. Arima Y, Iwata H (2007) Effect of wettability and surface functional groups on protein adsorption and cell adhesion using well-defined mixed self-assembled monolayers. Biomaterials 28(20):3074–3082

    CAS  PubMed  Google Scholar 

  61. Yun YJ et al (2019) Cellular organization of three germ layer cells on different types of noncovalent functionalized graphene substrates. Mater Sci Eng C 103(2018):109729

    CAS  Google Scholar 

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Acknowledgments

We would like to thank to Mehmet Yumak, Ahmet Turan Talaş, and all the employees of Boğaziçi University Life Science and Technologies Implementation and Research Center.

Funding

This work is supported by Boğaziçi University Research Fund by Grant No. 10040 and No. 7142 and partially by Grant No. 6701.

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Authors and Affiliations

  1. Institute of Biomedical Engineering, Bogazici University, Uskudar, Istanbul, Turkey

    Fatma Şimşek, Osman Melih Can, Bora Garipcan & Özgür Kocatürk

  2. Department of Biomedical Engineering, Faculty of Engineering and Natural Sciences, Bahcesehir University, Besiktas, Istanbul, Turkey

    Yekta Ülgen

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  1. Fatma Şimşek
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  2. Osman Melih Can
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  3. Bora Garipcan
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  4. Özgür Kocatürk
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Contributions

Conceptualization: F.S, B.G. Study design: F.S., O.M.C, B.G, Y.U, O.K. Funding acquisition: F.S, O.M.C. Experimental design: F.S, O.M.C, B.G, O.K, Y.U. Data collection and data analysis: F.S. Drafting of the manuscript: F.S. Editing and revision of the manuscript: F.S, B.G, Y.U. Approval of the final version of manuscript: F.S, O.M.C, B.G, O.K, Y.U.

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Correspondence to Fatma Şimşek.

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Şimşek, F., Can, O.M., Garipcan, B. et al. Characterization of carotid endothelial cell proliferation on Au, Au/GO, and Au/rGO surfaces by electrical impedance spectroscopy. Med Biol Eng Comput 58, 1431–1443 (2020). https://doi.org/10.1007/s11517-020-02166-0

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