Laura Fusco^1,2,3,Arianna Gazzi^1,4,Christopher Shuck²,Marco Orecchioni⁵,Sènan Mickael D'Almeida⁶,Darawan Rinchai³,Eiman Ahmed³,Leeat Ahmed⁷,Davide Bedognetti³,Yury Gogotsi²,Lucia Delogu¹

University of Padua¹,Drexel University²,Sidra Medicine³,University of Trieste⁴,La Jolla Institute for Allergy and Immunology⁵,Ecole Polytechnique Fédérale de Lausanne (EPFL)⁶,Weizmann Institute of Science⁷

Abstract

There is a critical unmet need to detect and image two-dimensional (2D) materials within single cells and tissues while surveying a high degree of information from single cells. Here, we propose a versatile multiplexed label-free single-cell detection strategy based on single-cell mass cytometry by time-of-flight (CyTOF) and ion-beam imaging by time-of-flight (MIBI-TOF). [1] This strategy, “Label-free sINgle-cell tracKing of 2D matErials by mass cytometry and MIBI-TOF Design” (LINKED), enables nanomaterial detection and simultaneous measurement of multiple cell and tissue features. As a proof of concept, we selected a set of 2D materials, transition metal carbides, nitrides, and carbonitrides (MXenes), to ensure mass detection within the cytometry range while avoiding overlap with more than 70 currently available tags, each able to survey multiple biological parameters. In addition, MXenes have received substantial attention for their promise as biomedical nanotools. [2-5] First, we demonstrated their detection and quantification in 15 primary human immune cell subpopulations. Together with the detection, mass cytometry is used to capture several biological aspects of MXenes, such as their biocompatibility and cytokine production after their uptake. In vivo biodistribution experiments using a mixture of MXenes in mice confirmed the versatility of the detection strategy and revealed MXene accumulation in the liver, blood, spleen, lungs, and relative immune cell subtypes. Finally, we applied MIBI-TOF to detect MXenes in different organs revealing their spatial distribution. The label-free detection of 2D materials by mass cytometry at the single-cell level, on multiple cell subpopulations, and in multiple organs simultaneously, is expected to enable exciting new opportunities in biomedicine. References: 1. Fusco L. et al. Immune Profiling and Multiplexed Label-Free Detection of 2D MXenes by Mass Cytometry and High-Dimensional Imaging. Advanced Materials, 2022, 2205154. 2. Gogotsi, Y. & Anasori, B. The Rise of MXenes. ACS Nano, 2019, 13, 8491. 3. Fusco L. et al. Graphene and other 2D materials: a multidisciplinary analysis to uncover the hidden potential as cancer theranostics. Theranostics, 2020, 10, 5435. 4. Unal M.A. et al. 2D MXenes with antiviral and immunomodulatory properties: a pilot study against SARS-CoV-2. Nano Today 2021, 38, 101136. 5. Gazzi A. et al. Photodynamic Therapy Based on Graphene and MXene in Cancer Theranostics. Front. Bioeng. Biotechnol., 2019, 7, 295.