The scope of application of carbon nanomaterials in biomedical, environmental and industrial fields is recently substantially increasing. Since in vitro toxicity testing is the first essential step for any commercial usage, it is crucial to have a reliable method to analyze the potentially harmful effects of carbon nanomaterials. Even though researchers already reported the interference of carbon nanomaterials with common toxicity assays, there is still, unfortunately, a large number of studies that neglect this fact. In this study, we investigated interference of four bio-promising carbon nanomaterials (graphene acid (GA), cyanographene (GCN), graphitic carbon nitride (g-C3N4) and carbon dots (QCDs)) in commonly used LIVE/DEAD assay. When a standard procedure was applied, materials caused various types of interference. While positively charged g-C3N4 and QCDs induced false results through the creation of free agglomerates and intrinsic fluorescence properties, negatively charged GA and GCN led to false signals due to the complex quenching effect of the fluorescent dye of a LIVE/DEAD kit. Thus, we developed a new approach using a specific gating strategy based on additional controls that successfully overcame all types of interference and lead to reliable results in LIVE/DEAD assay. We suggest that the newly developed procedure should be a mandatory tool for all in vitro flow cytometry assays of any class of carbon nanomaterials.

Department of Physical Chemistry Faculty of Science Palacký University Olomouc 17 Listopadu 12 1192 771 00 Olomouc Czech Republic

IT4Innovations National Supercomputing Center VŠB Technical University of Ostrava 17 listopadu 15 2172 708 00 Ostrava Czech Republic

Laboratory for Particles Biology Interactions Empa Swiss Federal Laboratories for Materials Science and Technology Lerchenfeldstrasse 5 9014 St Gallen Switzerland

Nanotechnology Centre Centre of Energy and Environmental Technologies VŠB Technical University of Ostrava 17 Listopadu 15 2172 708 00 Ostrava Poruba Czech Republic

Regional Centre of Advanced Technologies and Materials Czech Advanced Technology and Research Institute Palacký University Olomouc Šlechtitelů 27 779 00 Olomouc Czech Republic

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Li Z., Wang L., Li Y., Feng Y., Feng W. Carbon-based functional nanomaterials: Preparation, properties and applications. Compos. Sci. Technol. 2019;179:10–40. doi: 10.1016/j.compscitech.2019.04.028. DOI

Jaleel J.A., Pramod K. Artful and multifaceted applications of carbon dot in biomedicine. J. Control. Release. 2018;269:302–321. doi: 10.1016/j.jconrel.2017.11.027. PubMed DOI

Novoselov K.S., Geim A.K., Morozov S.V., Jiang D., Zhang Y., Dubonos S.V., Grigorieva I.V., Firsov A.A. Electric field effect in atomically thin carbon films. Science. 2004;306:666–669. doi: 10.1126/science.1102896. PubMed DOI

Gupta A., Sakthivel T., Seal S. Recent development in 2D materials beyond graphene. Prog. Mater. Sci. 2015;73:44–126. doi: 10.1016/j.pmatsci.2015.02.002. DOI

Georgakilas V., Tiwari J.N., Kemp K., Perrnan J.A., Bourlinos A.B., Kim K.S., Zboril R. Noncovalent Functionalization of Graphene and Graphene Oxide for Energy Materials, Biosensing, Catalytic, and Biomedical Applications. Chem. Rev. 2016;116:5464–5519. doi: 10.1021/acs.chemrev.5b00620. PubMed DOI

Zhu C., Du D., Lin Y. Graphene and graphene-like 2D materials for optical biosensing and bioimaging: A review. 2D Mater. 2015;2:032004. doi: 10.1088/2053-1583/2/3/032004. DOI

Bakandritsos A., Pykal M., Błoński P., Jakubec P., Chronopoulos D.D., Poláková K., Georgakilas V., Čépe K., Tomanec O., Ranc V., et al. Otyepka, Cyanographene and Graphene Acid: Emerging Derivatives Enabling High-Yield and Selective Functionalization of Graphene. ACS Nano. 2017;11:2982–2991. doi: 10.1021/acsnano.6b08449. PubMed DOI PMC

Liao G., He F., Li Q., Zhong L., Zhao R., Che H., Gao H., Fang B. Emerging graphitic carbon nitride-based materials for biomedical applications. Prog. Mater. Sci. 2020;112:100666. doi: 10.1016/j.pmatsci.2020.100666. DOI

Hola K., Zhang Y., Wang Y., Giannelis E.P., Zboril R., Rogach A.L. Carbon dots—Emerging light emitters for bioimaging, cancer therapy and optoelectronics. Nano Today. 2014;9:590–603. doi: 10.1016/j.nantod.2014.09.004. DOI

Wang A.-J., Li H., Huang H., Qian Z.-S., Feng J.-J. Fluorescent graphene-like carbon nitrides: Synthesis, properties and applications. J. Mater. Chem. C. 2016;4:8146–8160. doi: 10.1039/C6TC02330D. DOI

Devi P., Saini S., Kim K.-H. The advanced role of carbon quantum dots in nanomedical applications. Biosens. Bioelectron. 2019;141:111158. doi: 10.1016/j.bios.2019.02.059. PubMed DOI

Malina T., Poláková K., Skopalík J., Milotová V., Hola K., Havrdová M., Tománková K.B., Čmiel V., Sefc L., Zbořil R. Carbon dots for in vivo fluorescence imaging of adipose tissue-derived mesenchymal stromal cells. Carbon. 2019;152:434–443. doi: 10.1016/j.carbon.2019.05.061. DOI

Drasler B., Sayre P., Steinhäuser K.G., Fink A., Rothen-Rutishauser B. In vitro approaches to assess the hazard of nanomaterials. NanoImpact. 2017;8:99–116. doi: 10.1016/j.impact.2017.08.002. DOI

Krewski D., Acosta D., Andersen M., Anderson H., Bailar J.C., Boekelheide K., Brent R., Charnley G., Cheung V.G., Green S., et al. Toxicity Testing in the 21st Century: A Vision and a Strategy. J. Toxicol. Environ. Health Part B. 2010;13:51–138. doi: 10.1080/10937404.2010.483176. PubMed DOI PMC

Andersen M.E., Krewski D. Toxicity Testing in the 21st Century: Bringing the Vision to Life. Toxicol. Sci. 2008;107:324–330. doi: 10.1093/toxsci/kfn255. PubMed DOI

Romeo D., Salieri B., Hischier R., Nowack B., Wick P. An integrated pathway based on in vitro data for the human hazard assessment of nanomaterials. Environ. Int. 2020;137:105505. doi: 10.1016/j.envint.2020.105505. PubMed DOI

Kroll A., Pillukat M.H., Hahn D., Schnekenburger J. Interference of engineered nanoparticles with in vitro toxicity assays. Arch. Toxicol. 2012;86:1123–1136. doi: 10.1007/s00204-012-0837-z. PubMed DOI

Ong K.J., MacCormack T., Clark R.J., Ede J.D., Ortega V.A., Felix L., Dang M.K.M., Ma G., Fenniri H., Veinot J.G.C., et al. Widespread Nanoparticle-Assay Interference: Implications for Nanotoxicity Testing. PLoS ONE. 2014;9:e90650. doi: 10.1371/journal.pone.0090650. PubMed DOI PMC

Guadagnini R., Kenzaoui B.H., Walker L., Pojana G., Magdolenova Z., Bilanicova D., Saunders M., Juillerat-Jeanneret L., Marcomini A., Huk A. Boland, Toxicity screenings of nanomaterials: Challenges due to interference with assay processes and components of classic in vitro tests. Nanotoxicology. 2015;9:13–24. doi: 10.3109/17435390.2013.829590. PubMed DOI

Andraos C., Yu I.J., Gulumian M. Interference: A Much-Neglected Aspect in High-Throughput Screening of Nanoparticles. Int. J. Toxicol. 2020;39:397–421. doi: 10.1177/1091581820938335. PubMed DOI

Labouta H., Asgarian N., Rinker K., Cramb D.T. Meta-Analysis of Nanoparticle Cytotoxicity via Data-Mining the Literature. ACS Nano. 2019;13:1583–1594. doi: 10.1021/acsnano.8b07562. PubMed DOI

Wright P.C., Qin H., Choi M.M., Chiu N.H., Jia Z. Carbon nanodots interference with lactate dehydrogenase assay in human monocyte THP-1 cells. SpringerPlus. 2014;3:615. doi: 10.1186/2193-1801-3-615. PubMed DOI PMC

Casey A., Herzog E., Davoren M., Lyng F., Byrne H., Chambers G. Spectroscopic analysis confirms the interactions between single walled carbon nanotubes and various dyes commonly used to assess cytotoxicity. Carbon. 2007;45:1425–1432. doi: 10.1016/j.carbon.2007.03.033. DOI

Monteiro-Riviere N., Inman A.O. Challenges for assessing carbon nanomaterial toxicity to the skin. Carbon. 2006;44:1070–1078. doi: 10.1016/j.carbon.2005.11.004. DOI

Wörle-Knirsch J.M., Pulskamp A.K., Krug H.F., Wörle-Knirsch J.M., Pulskamp A.K., Krug H.F. Oops They Did It Again! Carbon Nanotubes Hoax Scientists in Viability Assays. Nano Lett. 2006;6:1261–1268. doi: 10.1021/nl060177c. PubMed DOI

Holder A., Goth-Goldstein R., Lucas D., Koshland C.P. Particle-Induced Artifacts in the MTT and LDH Viability Assays. Chem. Res. Toxicol. 2012;25:1885–1892. doi: 10.1021/tx3001708. PubMed DOI PMC

Belyanskaya L., Manser P., Spohn P., Bruinink A., Wick P. The reliability and limits of the MTT reduction assay for carbon nanotubes-cell interaction. Carbon. 2007;45:2643–2648. doi: 10.1016/j.carbon.2007.08.010. DOI

Tuchin V.V., Tárnok A., Zharov V.P. In vivo flow cytometry: A horizon of opportunities. Cytom. Part A. 2011;79:737–745. doi: 10.1002/cyto.a.21143. PubMed DOI PMC

Bakke A.C. The Principles of Flow Cytometry. Lab. Med. 2001;32:207–211. doi: 10.1309/2H43-5EC2-K22U-YC6T. DOI

Bohmer N., Rippl A., May S., Walter A., Heo M.B., Kwak M., Roesslein M., Song N.W., Wick P., Hirsch C. Interference of engineered nanomaterials in flow cytometry: A case study. Colloids Surf. B: Biointerfaces. 2018;172:635–645. doi: 10.1016/j.colsurfb.2018.09.021. PubMed DOI

Svoboda L., Praus P., de Lima M.J.B.P., Sampaio M.J., Matýsek D., Ritz M., Dvorský R., Faria J.L., Silva C.G. Graphitic carbon nitride nanosheets as highly efficient photocatalysts for phenol degradation under high-power visible LED irradiation. Mater. Res. Bull. 2018;100:322–332. doi: 10.1016/j.materresbull.2017.12.049. DOI

Svoboda L., Škuta R., Matějka V., Dvorský R., Matýsek D., Henych J., Mančík P., Praus P. Graphene oxide and graphitic carbon nitride nanocomposites assembled by electrostatic attraction forces: Synthesis and characterization. Mater. Chem. Phys. 2019;228:228–236. doi: 10.1016/j.matchemphys.2019.02.077. DOI

Monopoli M.P., Åberg C., Salvati A., Dawson K.A. Biomolecular coronas provide the biological identity of nanosized materials. Nat. Nanotechnol. 2012;7:779–786. doi: 10.1038/nnano.2012.207. PubMed DOI

Patil S., Sandberg A., Heckert E., Self W., Seal S. Protein adsorption and cellular uptake of cerium oxide nanoparticles as a function of zeta potential. Biomaterials. 2007;28:4600–4607. doi: 10.1016/j.biomaterials.2007.07.029. PubMed DOI PMC

Walkey C.D., Chan W.C.W. Understanding and controlling the interaction of nanomaterials with proteins in a physiological environment. Chem. Soc. Rev. 2012;41:2780–2799. doi: 10.1039/C1CS15233E. PubMed DOI

Doorley G.W., Payne C.K. Cellular binding of nanoparticles in the presence of serum proteins. Chem. Commun. 2011;47:466–468. doi: 10.1039/C0CC02618B. PubMed DOI

Glancy D., Zhang Y., Wu J.L., Ouyang B., Ohta S., Chan W.C. Characterizing the protein corona of sub-10 nm nanoparticles. J. Control. Release. 2019;304:102–110. doi: 10.1016/j.jconrel.2019.04.023. PubMed DOI

Longin C., Petitgonnet C., Guilloux-Benatier M., Rousseaux S., Alexandre H. Application of flow cytometry to wine microorganisms. Food Microbiol. 2017;62:221–231. doi: 10.1016/j.fm.2016.10.023. PubMed DOI