In hundreds of articles published over the past two decades, nanoparticles have been described as probes for sensing and imaging of a variety of intracellular cytosolic targets. However, nanoparticles generally enter cells by endocytosis with only a small fraction reaching the cytosol. Most of those articles do not describe the mechanisms of nanoparticles entry into the cell and therefore the paradox of sensing a target in the cytosol when most particles do not access that compartment remains. To address this paradox, we are initiating a replication project of some of the most influential articles in this field with the aim of confirming their ability to detect their targets and getting additional insights into their intracellular localisation. Thus, this article is a pre-registered report for the first replication of this project, namely the replication of Zhu et al ‘Carbon‐Dot‐Based Dual‐Emission Nanohybrid Produces a Ratiometric Fluorescent Sensor for In Vivo Imaging of Cellular Copper Ions’. To achieve our aim, we will carry out both a direct replication of the experiments in the original article, with the addition of some experiments and controls, and a conceptual replication where we test the claim that this ratiometric probe is able to sense intracellular copper levels in a more biologically relevant system. We hope that this study will contribute to a better understanding of the intracellular fate of nanoparticles and, with the help of the scientific community, help set standards in the design and reporting of studies in this field.
This study presents the results of a replication effort aimed at reproducing key findings from the 2012 article ‘Carbon-dot-based dual-emission nanohybrid produces a ratiometric fluorescent sensor for in vivo imaging of cellular copper ions’ by Zhu et al. The original study claimed that carbon quantum dots (CQDs) functionalized with N-(2-aminoethyl)-N,N′,N′-tris(pyridine-2- ylmethyl)ethane-1,2-diamine (AE-TPEA) (CQDs-TPEA) and their hybrid with CdSe/ZnS quantum dots (CdSe@C-TPEA) exhibited ratiometric fluorescence quenching upon exposure to Cu2+ ions, enabling their application as a dual-emission sensor for intracellular Cu2+ imaging. Despite closely following the described synthesis and characterization protocols, we were unable to reproduce the Cu2+-induced fluorescence quenching, undermining the primary claim of the original study and making it impossible to pursue the investigation in live cells. Furthermore, upon close examination of the original data, concerns regarding spectral anomalies and potential inconsistencies were raised, prompting us to abort the replication study. This Registered Report has been officially endorsed by Peer Community In Registered Reports: https://doi.org/10.24072/pci.rr.101070.